A SURF4-to-proteoglycan relay mechanism that mediates the sorting and secretion of a tagged variant of sonic hedgehog

Sonic Hedgehog (Shh) is a key signaling molecule that plays important roles in various developmental processes in mammals. Although the signal transduction pathway activated by Shh is well understood, the regulation of its secretion remains unclear. Newly synthesized Shh is imported into the endoplasmic reticulum (ER), where it undergoes a series of posttranslational modifications to produce the mature lipid-modified amino-terminal fragment. Here, we have analyzed the molecular mechanisms that mediate secretion of the N-terminal fragment of Shh (ShhN). We found that the Cardin–Weintraub (CW) motif in Shh is necessary and sufficient for ER-to-Golgi transport of ShhN. Mechanistic analyses revealed that a cargo receptor, Surfeit locus protein 4 (SURF4), interacts directly with the CW motif of ShhN to regulate packaging of ShhN into COPII vesicles. ShhN and SURF4 interact with each other at the ER and separate from each other after entering the Golgi. The CW motif is known to interact with proteoglycans (PGs) that are predominantly synthesized at the Golgi. Interestingly, we found that PGs compete with SURF4 to bind ShhN and that inhibiting synthesis of PGs causes defects in export of ShhN from the trans Golgi network (TGN). SURF4 and PG maturation are also important for intracellular traffic of full length Shh in mammalian cells. Our study suggests a SURF4-to-PG relay mechanism that mediates the sorting and secretion of Shh, providing insight into the biosynthetic trafficking of Shh.

The Hedgehog (Hh) signaling pathway plays an important role in various developmental processes in metazoans (1, 2). Mutations of key components that regulate Hh signaling are associated with many human diseases (3). Hh was first found in the Drosophila larval epidermis. It mediates larval segment development and adult appendage patterning (4). In mammals, there are three Hh-family members, Sonic hedgehog (Shh), Indian hedgehog (Ihh), and Desert hedgehog (Dhh). Ihh regulates the proliferation and differentiation of chondrocytes (5). Dhh functions in gonads, regulating testis organogenesis, spermatogenesis (6, 7), and follicle development in the ovary (8). Shh functions more extensively than the other two Hh members: it regulates embryonic patterning (4), specification of cell types in the nervous system (9), axon guidance (10), cell differentiation, and organ development (11).Hh is synthesized as a full-length precursor Hh (HhFL). After entering the endoplasmic reticulum (ER), HhFL is autocleaved into two parts: an N-terminal Hedge domain (HhN) and a C-terminal Hog domain (HhC) (1). HhC is degraded through ER-associated degradation (12). HhN undergoes lipid modifications, in which a cholesterol molecule is covalently linked to the C terminus and a palmitoyl group is linked to the N terminus (13–15). Lipid-modified HhN subsequently exits the ER and is delivered via the secretory pathway to the plasma membrane. Once at the plasma membrane, Hh is released into the extracellular matrix and ultimately recognized by its receptors on the plasma membrane of target cells to induce downstream signal transduction.Although significant progress has been achieved in understanding the Hh signaling pathway in target cells, the molecular mechanisms that mediate secretion of newly synthesized Shh proteins from the producing cells are still unclear. The ER is the first station where newly synthesized proteins enter the secretory pathway. In this compartment, cargo proteins are generally recognized by the coat protein complex II (COPII) to be packaged into vesicles and exported from the ER. Soluble cargo proteins in the ER lumen cannot directly engage the COPII coat but instead are captured into vesicles by transmembrane cargo receptors. One mammalian cargo receptor, ERGIC53, is a mannose-specific lectin that recognizes N-linked glycoproteins in the ER lumen (16, 17). The p24 family of proteins function as cargo receptors to regulate ER export of glycosylphosphatidylinositol (GPI)-anchored proteins (18). Mammalian orthologs of yeast ER vesicle (Erv) proteins have also been thought to function as cargo receptors (16). Surfeit locus protein 4 (SURF4), the mammalian ortholog of Erv29p, regulates ER export of soluble proteins, including lipoproteins and proprotein convertase subtilisin/kexin type 9 (PCSK9) (19–21). SURF4 recognizes amino-terminal tripeptide motifs of soluble cargo proteins and participates in ER exit site (ERES) organization (19, 22). The cargo receptors that mediate sorting of Shh in the secretory pathway remain unknown.Here, we examined trafficking of the N-terminal fragment of Shh without the cholesterol modification (referred to as ShhN). We utilized the Retention Using Selective Hook (RUSH) assay (23) to analyze the kinetics of trafficking of ShhN along the secretory pathway. We reconstituted the packaging of ShhN into transport vesicles in vitro and utilized this assay to quantitatively measure packaging efficiency. Our study reveals cellular factors and underlying mechanisms that mediate the sorting and secretion of Shh, providing insight into the biosynthetic trafficking of Shh.     

Phosphoregulatory protein 14-3-3 facilitates SAC1 transport from the endoplasmic reticulum

Most secretory cargo proteins in eukaryotes are synthesized in the endoplasmic reticulum and actively exported in membrane-bound vesicles that are formed by the cytosolic coat protein complex II (COPII). COPII proteins are assisted by a variety of cargo-specific adaptor proteins required for the concentration and export of secretory proteins from the endoplasmic reticulum (ER). Adaptor proteins are key regulators of cargo export, and defects in their function may result in disease phenotypes in mammals. Here we report the role of 14-3-3 proteins as a cytosolic adaptor in mediating SAC1 transport in COPII-coated vesicles. Sac1 is a phosphatidyl inositol-4 phosphate (PI4P) lipid phosphatase that undergoes serum dependent translocation between the endoplasmic reticulum and Golgi complex and controls cellular PI4P lipid levels. We developed a cell-free COPII vesicle budding reaction to examine SAC1 exit from the ER that requires COPII and at least one additional cytosolic factor, the 14-3-3 protein. Recombinant 14-3-3 protein stimulates the packaging of SAC1 into COPII vesicles and the sorting subunit of COPII, Sec24, interacts with 14-3-3. We identified a minimal sorting motif of SAC1 that is important for 14-3-3 binding and which controls SAC1 export from the ER. This LS motif is part of a 7-aa stretch, RLSNTSP, which is similar to the consensus 14-3-3 binding sequence. Homology models, based on the SAC1 structure from yeast, predict this region to be in the exposed exterior of the protein. Our data suggest a model in which the 14-3-3 protein mediates SAC1 traffic from the ER through direct interaction with a sorting signal and COPII.Most of the transmembrane secretory cargo proteins from the endoplasmic reticulum (ER) are selectively exported in cytosolic coat protein complex II (COPII) vesicles via direct interaction of their export motif with the COPII coat. The COPII coat core machinery consists of five cytosolic proteins: Sar1, Sec23, Sec24, Sec13, and Sec31 (secretory pathway proteins) (1). Sec24 is considered to be the primary subunit responsible for binding to membrane cargo proteins at the ER and concentrating them into the forming vesicle (2). Some of these cargo proteins require the assistance of cytosolic or membrane-spanning accessory adaptor proteins for their incorporation into COPII vesicles. Several adaptor proteins have been identified to assist the COPII machinery in yeast (3–5); however, fewer have been characterized in higher eukaryotes. In metazoans, ERGIC-53 mediates the export of blood clotting factors, Cathepsin Z and C and α-1 antitrypsin (6), and SCAP [sterol-regulatory elementary binding protein (SREBP) cleavage activating protein] mediates the regulated transport of SREBP protein from the ER to the Golgi in cells that are sterol-deficient (7). Most COPII adaptor proteins are membrane-embedded, but at least one example of a cytosolic accessory protein, 14-3-3, has been proposed to control the anterograde trafficking of many of cell surface receptor proteins, possibly at the level of the ER (8). 14-3-3s are small (30 kDa), acidic, and ubiquitously expressed eukaryotic proteins that are conserved from yeast to mammals and modulate various cellular processes by interacting with a variety of target proteins (9, 10). These include cell cycle regulation, signaling by MAP kinases, apoptosis, and transfer of signaling molecules between the nucleus and cytosol (11–14). Yeast cell viability depends on the expression of at least one of the two 14-3-3 isoforms (Bmh1 and Bmh2) (15). There are seven different isoforms in mammals (β, γ, δ, ε, η, σ, θ), some of which show differential tissue localization (14). Because of their redundant roles in cellular processes, depleting cellular levels of 14-3-3 to study a particular process poses a challenge. It is thought that their role in trafficking is to interfere with the ER retention/retrieval motif of target membrane proteins, and thus promote the transport of these cargos to the cell surface (16). For some proteins (e.g., KCNK3 and MHC class II, GPR15) (17–19), recruitment of 14-3-3 requires phosphorylation of a residue involved in 14-3-3 binding, whereas in other proteins (e.g., Kir6.2) 14-3-3 recognizes the correct assembly of multimeric proteins (20, 21).In this paper we examine the role of 14-3-3 proteins as an adaptor for COPII vesicular transport of SAC1 (suppressor of actin mutations 1-like protein). SAC1 is a phosphatidyl inositol-4 (PI4) lipid phosphatase that belongs to a family of enzymes with a CX₅R(T/S) Sac catalytic domain, which is conserved from yeast to metazoans. Sac proteins control several cellular processes, including phosphoinositide homeostasis, membrane trafficking, and cytoskeleton organization. SAC1 is a 587-aa transmembrane protein with both N- and C-terminal domains exposed to the cytosol. Deletion of SAC1 in yeast and mammalian cells leads to changes in Golgi morphology and function and a SAC1 mouse knockout is embryonically lethal. Recently, SAC1 has been identified as Drosophila vesicle-associated protein binding partner and down-regulation of Drosophila vesicle-associated protein or SAC1 in Drosophila leads to the pathogenesis associated with amyotrophic lateral sclerosis (22).It has been reported previously that SAC1 is localized to the Golgi membranes only when cells are starved for nutrients or growth factors, but remains in the ER under normal growth conditions (23, 24). Given the role for PI(4)P in vesicle traffic from the trans Golgi network, starvation conditions that lodge SAC1 and thus deplete the local supply of PI(4)P in the Golgi may suppress anterograde traffic in cells that must cease net cell growth. The regulation of SAC1 traffic may be crucial to the control of cell growth and anterograde membrane traffic.The retrieval of mammalian SAC1 from the Golgi to the ER in the presence of growth factors or mitogens is controlled by COPI-mediated retrograde transport and requires the p38 MAPK pathway (23). Although the regulation of SAC1 retrieval from the Golgi has been reported, little is known about the control of SAC1 export from the ER under conditions of serum starvation. Recently, the N-terminal cytoplasmic domain of SAC1 was reported to contribute to Golgi localization in mammalian cells (25). We have established a cell-free reconstitution system that recapitulates the biogenesis and ER export of SAC1 and identified 14-3-3 proteins as an important factor in the packaging of SAC1 into COPII transport vesicles. Given the role of 14-3-3 proteins in various signaling pathways and the fact that SAC1 transport is affected by the p38 MAPK pathway, an understanding of the molecular role of 14-3-3 proteins in vesicular traffic could provide a mechanistic link between signaling and membrane assembly (23).     

From the Cover: Membrane raft association is a determinant of plasma membrane localization

The lipid raft hypothesis proposes lateral domains driven by preferential interactions between sterols, sphingolipids, and specific proteins as a central mechanism for the regulation of membrane structure and function; however, experimental limitations in defining raft composition and properties have prevented unequivocal demonstration of their functional relevance. Here, we establish a quantitative, functional relationship between raft association and subcellular protein sorting. By systematic mutation of the transmembrane and juxtamembrane domains of a model transmembrane protein, linker for activation of T-cells (LAT), we generated a panel of variants possessing a range of raft affinities. These mutations revealed palmitoylation, transmembrane domain length, and transmembrane sequence to be critical determinants of membrane raft association. Moreover, plasma membrane (PM) localization was strictly dependent on raft partitioning across the entire panel of unrelated mutants, suggesting that raft association is necessary and sufficient for PM sorting of LAT. Abrogation of raft partitioning led to mistargeting to late endosomes/lysosomes because of a failure to recycle from early endosomes. These findings identify structural determinants of raft association and validate lipid-driven domain formation as a mechanism for endosomal protein sorting.Recent advances in superresolution microscopy (1), lipid analysis (2, 3), and plasma membrane (PM) isolation (4, 5) have confirmed the coexistence of lipid-driven, fluid domains in biological membranes. The relatively ordered domains, known as “membrane rafts,” have been proposed to be involved in protein sorting (6), viral/pathogen trafficking (3, 7), and PM signaling in a variety of contexts (8). However, despite the increasing evidence confirming the existence of dynamic, nanoscopic membrane rafts, the functional consequences of this phenomenon remain speculative because of the limitations of the previously used methods for defining raft association, i.e., the resistance of membrane components to solubilization by nonionic detergents (9).Lipid-mediated domains have been implicated as a mechanism for protein sorting in the latter stages of the secretory pathway (trans-Golgi network to the PM) (2, 6, 10–12), with analogous pathways mediating endosomal sorting/recycling (13, 14). Raft lipids (i.e., sterols and sphingolipids) are significantly enriched at the PM (15–17), and recent observations confirm that these lipids also are enriched in sorting vesicles destined for the PM (2, 11). For proteins, several specific cytosolic signals exist for adapter/coat-mediated sorting between cellular organelles (18); in parallel, protein–lipid interactions through hydrophobic transmembrane domains (TMDs) also have been shown to regulate trafficking. For example, a strong correlation exists between the TMD length of bitopic proteins and their organelle specificity (19, 20), with longer TMDs targeting proteins to the PM and shorter TMDs found in the endoplasmic reticulum (ER), Golgi apparatus, and endocytic organelles. These findings suggest cargo sorting in the secretory and endocytic pathways, with proteins containing longer TMDs, together with sphingolipids and cholesterol, being specifically trafficked to the PM, although the mechanism for this observation remains unresolved.One possibility for sorting of specific lipid classes along with proteins containing longer TMDs is lateral segregation and coalescence of ordered domains, followed by either domain-induced (21) or cytoskeleton-assisted (22) budding of raft-enriched transport vesicles. Proteins using this “raft pathway” would not require cytosolic sorting signals but rather would be recruited to transport vesicles by their raft affinity, i.e., their propensity to interact with specific lipids, ordered domains, or other raft-embedded proteins. Because ordered phases in lipid model systems consistently have been shown to be 0.6–1.5 nm thicker than disordered domains (23, 24), raft-associated transmembrane (TM) proteins would be predicted to have longer TMDs. TMD length-dependent protein sorting between coexisting lipid domains has been addressed experimentally only recently by measuring partitioning of an oligomeric toxin (perfringolysin O) with multiple (35–40) TM segments in synthetic, phase-separated liposomes (25). Whether these observations extend to single-pass TM proteins in biological membranes is unknown.To evaluate the role of lipid-driven raft domains as a mechanism for subcellular protein sorting, we quantitatively compared the raft association of 30 TM protein variants with their subcellular localization. To quantify raft partitioning of the constructs comprising single-pass TM proteins with varying TMD lengths and sequences, we used giant PM vesicles (GPMVs). GPMVs are cell-detached PM blebs whose protein (26) and lipid (27) diversity mirrors that of the native PM. These PM vesicles separate into coexisting liquid phases (4) with different order (28), which recruit membrane components in accordance with their predicted raft affinity, i.e., saturated lipids, glycosphingolipids (29), glycosylphosphatidyl inositol-anchored proteins (4), and palmitoylated proteins (30) partition to the ordered phase, denoted here as the “raft phase.” Most importantly, these vesicles provide a platform for repeatable, direct, and quantitative analysis of raft partitioning (30), allowing investigation of the structural determinants of raft association and its effect on protein function. We find that perturbation of raft partitioning by three independent means (decreasing TMD length, mutation of palmitoylation sites, and TMD sequence manipulation) perturbed subcellular localization, leading to missorting of PM proteins to late endosomes and lysosomes because of a failure to recycle nonraft proteins from early endosomes (EEs). These results confirm the presence of a raft-mediated recycling route in nonpolarized cells, begin to define the molecular parameters for protein association with raft domains, and suggest an explanation for the accumulation of proteins with longer TMDs at the PM.     

From the Cover: PNAS Plus: Arl1p regulates spatial membrane organization at the trans-Golgi network through interaction with Arf-GEF Gea2p and flippase Drs2p

ADP ribosylation factors (Arfs) are the central regulators of vesicle trafficking from the Golgi complex. Activated Arfs facilitate vesicle formation through stimulating coat assembly, activating lipid-modifying enzymes and recruiting tethers and other effectors. Lipid translocases (flippases) have been implicated in vesicle formation through the generation of membrane curvature. Although there is no evidence that Arfs directly regulate flippase activity, an Arf-guanine-nucleotide-exchange factor (GEF) Gea2p has been shown to bind to and stimulate the activity of the flippase Drs2p. Here, we provide evidence for the interaction and activation of Drs2p by Arf-like protein Arl1p in yeast. We observed that Arl1p, Drs2p and Gea2p form a complex through direct interaction with each other, and each interaction is necessary for the stability of the complex and is indispensable for flippase activity. Furthermore, we show that this Arl1p-Drs2p-Gea2p complex is specifically required for recruiting golgin Imh1p to the Golgi. Our results demonstrate that activated Arl1p can promote the spatial modulation of membrane organization at the trans-Golgi network through interacting with the effectors Gea2p and Drs2p.ADP ribosylation factors (Arf)/SARs, a subfamily of the Ras small GTP-binding protein superfamily, are critical regulators of vesicular trafficking in eukaryotic cells (1–3). Like Ras, Arf and Arf-like (Arl) proteins cycle between the inactive GDP-bound form and active GTP-bound forms to carry out their functions. The conversion from the GDP-bound to the GTP-bound form is facilitated by Arf guanine-nucleotide-exchange factor (Arf-GEF), whereas GTP hydrolysis is catalyzed by an Arf GTPase-activating protein (Arf-GAP) (4, 5). Activated Arf recruits numerous proteins to the membrane or activates lipid-modifying enzymes to facilitate vesicle formation, including vesicle coat assembly, membrane curvature generation, lipid composition alteration, and final membrane fission (4, 6–8).Arl1p is the best-studied Arl protein and is expressed in organisms ranging from yeast to humans (9, 10). Both yeast Arl1p and human Arl1 localize to the trans-Golgi network (TGN), where they regulate multiple membrane-trafficking pathways (9, 11, 12). Yeast Arl1p participates in three different pathways at the TGN, including glycosylphosphatidylinositol (GPI)-anchored protein transport, clathrin adaptor protein Gga recruitment, and targeting of the GRIP domain–containing protein Imh1p to the Golgi. In addition to Imh1p, the Arf-GEF–like protein, Mon2p (also called Ysl2p), and the clathrin adaptor protein, Gga2p, directly interact with Arl1p and are considered to be Arl1p effectors (13).Arf-GEFs catalyze the GTP binding of Arf/Arl through the conserved Sec7 domain and are upstream regulators of Arf/Arl activities (14, 15). In yeast, the human GBF1 homolog Gea2p, which functions redundantly with Gea1p, displays Arf-GEF activity toward Arf1p and is believed to be involved in ER-Golgi and intra-Golgi transport (16, 17). Recently, Gea2p was shown to directly interact with the TGN-localized aminophospholipid translocase Drs2p and to stimulate its flippase activity (18, 19). Similarly, it was reported that the Arf-GEF–like protein Mon2p interacted with both Arl1p and the Drs2p family protein Neo1p and that the Mon2-Neo1-Arl1 complex cooperated to regulate the Golgi recruitment of Gga2p (13). These results suggest that Arf-GEFs may act with members of the Drs2p subfamily of P-type ATPases to facilitate lipid translocation. However, the role of Gea2p in Drs2p-mediated flippase activity is not clear.Flippases translocate specific phospholipid molecules to establish the asymmetry of membranes (19–22). Therefore, flippases can drive membrane bending toward the cytosol and contribute to vesicle-mediated protein transport by facilitating the generation of membrane curvature that is inherent to this process (23). The inactivation of Drs2p results in defects in the formation of an Arf/clathrin-dependent class of post-Golgi vesicles that carry exocytic cargo (24). Moreover, a mutation in DRS2 was shown to cause synthetic lethality with clathrin heavy chain and ARF1 (25). These observations indicate the importance of flippase activity for vesicle trafficking, and Drs2p is involved in protein transport at the TGN.To explore the possibility that Arl1p might, like Arf proteins, play a role in regulating membrane dynamics, we use an analysis of both physical interaction and functional interaction to demonstrate that Drs2p and Gea2p are effectors of Arl1p. We find that these proteins—Arl1p, Drs2p, and Gea2p—are all required to form a stable complex to stimulate Drs2p flippase activity and complete certain functions of Arl1p. Our study provides insight into the molecular mechanisms of spatial membrane dynamics at the TGN and their regulation by the Arl1p-Gea2p-Drs2p cooperating network.     

The EM structure of the TRAPPIII complex leads to the identification of a requirement for COPII vesicles on the macroautophagy pathway

The transport protein particle (TRAPP) III complex, comprising the TRAPPI complex and additional subunit Trs85, is an autophagy-specific guanine nucleotide exchange factor for the Rab GTPase Ypt1 that is recruited to the phagophore assembly site when macroautophagy is induced. We present the single-particle electron microscopy structure of TRAPPIII, which reveals that the dome-shaped Trs85 subunit associates primarily with the Trs20 subunit of TRAPPI. We further demonstrate that TRAPPIII binds the coat protein complex (COP) II coat subunit Sec23. The COPII coat facilitates the budding and targeting of ER-derived vesicles with their acceptor compartment. We provide evidence that COPII-coated vesicles and the ER-Golgi fusion machinery are needed for macroautophagy. Our results imply that TRAPPIII binds to COPII vesicles at the phagophore assembly site and that COPII vesicles may provide one of the membrane sources used in autophagosome formation. These events are conserved in yeast to mammals.Macroautophagy is a highly conserved catabolic process that uses a specialized membrane trafficking pathway to target proteins and organelles for degradation (1). Defects in this process have been linked to a variety of human diseases, including neurodegenerative diseases such as Parkinson’s disease (2). Macroautophagy is induced by a variety of physiological stresses and begins with the expansion of a cup-shaped nucleating membrane called the phagophore, or isolation membrane. As the phagophore expands, it engulfs intracellular proteins and membranes that are marked for degradation. This expanding membrane eventually closes to become an autophagosome, a double-membrane structure that seals its contents from the cytosol and delivers it to the lysosome or vacuole for degradation. A central unanswered question in the autophagy field is the mechanism by which the phagophore forms and matures into an autophagosome. Although it was once thought that the phagophore assembles de novo, recent evidence suggests it forms from a preexisting compartment. Compartments on the secretory pathway, including the endoplasmatic reticulum (ER) and Golgi complex, have been invoked in phagophore assembly (3, 4).A collection of ATG (autophagy-related) genes, the products of which regulate autophagy, were identified in the yeast Saccharomyces cerevisiae (1). Many of the Atg proteins needed for macroautophagy in yeast are shared with the biosynthetic cytoplasm to vacuole targeting (Cvt) pathway that transports certain hydrolases into the vacuole. Both pathways require the sequestration of cargo within a double-membrane structure; however, only the macroautophagy pathway is conserved in higher eukaryotes (5). When autophagy is induced, ATG gene products assemble at the phagophore assembly site (PAS) in a hierarchical manner. The scaffold protein complex that organizes this site is the Atg17 complex (6, 7).Previous studies have shown that the transport protein particle (TRAPP) III complex, an autophagy-specic guanine nucleotide exchange factor (GEF) for the Rab GTPase Ypt1, is recruited to the PAS (8) by Atg17 (9). At the PAS, TRAPPIII activates Ypt1 (8), which then recruits its downstream effector, the serine/threonine Atg1 kinase, to the PAS (9). TRAPPIII is one of three multimeric GEFs, called TRAPPI, TRAPPII, and TRAPPIII, that activate Ypt1 on different trafficking pathways (10). TRAPPI, an elongated complex ∼18 nm in length (11, 12), binds to ER-derived COPII-coated vesicles via an interaction between the TRAPPI subunit Bet3 and the coat subunit Sec23 (11, 13). The TRAPPIII complex contains the same six subunits present in TRAPPI plus one unique subunit, Trs85, that targets this complex to the PAS (8, 9). Here, we describe the single-particle electron microscopy (EM) structure of TRAPPIII from S. cerevisiae. TRAPPIII (23 nm) is longer than TRAPPI with Trs85 capping one end of the complex. As in TRAPPI, the two Bet3 subunits in TRAPPIII are solvent accessible and are available for interaction with Sec23. This observation suggests that TRAPPIII may play a role in tethering COPII-coated vesicles at the PAS. Consistent with this proposal, we find that COPII vesicles accumulate at the PAS when autophagy is blocked. Additionally, we show that mutations in components of the ER-Golgi trafficking machinery, which mediate COPII vesicle fusion, disrupt autophagy. Finally, we find that COPII vesicles accumulate at or near ER-mitochondria contact sites when COS-7 cells are starved. ER-mitochondria contact sites are where autophagosomes form in mammalian cells (14).     

A mechanism for retromer endosomal coat complex assembly with cargo

Retromer is an evolutionarily conserved protein complex composed of the VPS26, VPS29, and VPS35 proteins that selects and packages cargo proteins into transport carriers that export cargo from the endosome. The mechanisms by which retromer is recruited to the endosome and captures cargo are unknown. We show that membrane recruitment of retromer is mediated by bivalent recognition of an effector of PI3K, SNX3, and the RAB7A GTPase, by the VPS35 retromer subunit. These bivalent interactions prime retromer to capture integral membrane cargo, which enhances membrane association of retromer and initiates cargo sorting. The role of RAB7A is severely impaired by a mutation, K157N, that causes Charcot–Marie–Tooth neuropathy 2B. The results elucidate minimal requirements for retromer assembly on the endosome membrane and reveal how PI3K and RAB signaling are coupled to initiate retromer-mediated cargo export.Sorting of cargo within the endosome determines whether it will be retained and ultimately degraded via lysosome-mediated turnover, or exported via a plasma membrane recycling or retrograde pathway that directs cargo to the TGN or recycling endosome. Genetic dissection of endosomal retrograde pathways in budding yeast (Saccharomyces cerevisiae) led to the identification of an endosome-associated protein complex termed retromer, composed of a Vps5–Vps17 heterodimer and a trimeric complex of the Vps26, Vps29, and Vps35 proteins (1). The retromer trimer, also called the “cargo recognition complex,” is the core functional unit of retromer, serving as a platform for recruiting many other factors to the endosome (2), and we shall refer herein to the trimer as retromer. It is now appreciated that retromer constitutes an ancient, evolutionarily conserved protein sorting complex that operates in multiple endosomal cargo export pathways (2, 3). Hence, elucidating the molecular mechanisms that underlie retromer function is key for understanding the endosomal system.The formation of a vesicular transport carrier is typically initiated by a GTPase module that elicits recruitment of a coat protein from the cytosol to a particular site on the membrane. Retromer is an effector of RAB7A [henceforth referred to as RAB7 (human) or Ypt7 (yeast)], a GTPase regulator of endosome dynamics and depletion of RAB7-GTP in cells results in a substantial loss of endosome-associated retromer (4–9). In addition to GTPase signaling modules, interactions of coat proteins with membrane lipids, such as phosphoinositides, contribute to coat assembly by increasing the avidity of membrane binding. There is no evidence that retromer directly recognizes membrane lipids (10, 11). Instead, retromer membrane recruitment is attributed to its association with any of several different sorting nexins (3), which are peripheral membrane proteins defined by the presence of a Phox homology (PX) domain that recognizes phosphatidylinositol 3-phosphate (PtdIns3P), a signature component of endosomal membranes. However, a formal test of this hypothesis is lacking. Retromer binds sorting nexin 3 [henceforth referred to as SNX3 (human) or Snx3 (yeast)] (12–15), and in SNX3 knockdown cells, less retromer is associated with endosomes (14). In this study, we show that SNX3 and RAB7 are coordinately recognized by retromer and that these interactions are sufficient to recruit retromer to a membrane where they poise retromer to capture integral membrane retrograde cargo.     

Structural insights into how vacuolar sorting receptors recognize the sorting determinants of seed storage proteins

In Arabidopsis, vacuolar sorting receptor isoform 1 (VSR1) sorts 12S globulins to the protein storage vacuoles during seed development. Vacuolar sorting is mediated by specific protein–protein interactions between VSR1 and the vacuolar sorting determinant located at the C terminus (ctVSD) on the cargo proteins. Here, we determined the crystal structure of the protease-associated domain of VSR1 (VSR1-PA) in complex with the C-terminal pentapeptide (₄₆₈RVAAA₄₇₂) of cruciferin 1, an isoform of 12S globulins. The ₄₆₈RVA₄₇₀ motif forms a parallel β-sheet with the switch III residues (₁₂₇TMD₁₂₉) of VSR1-PA, and the ₄₇₁AA₄₇₂ motif docks to a cradle formed by the cargo-binding loop (₉₅RGDCYF₁₀₀), making a hydrophobic interaction with Tyr99. The C-terminal carboxyl group of the ctVSD is recognized by forming salt bridges with Arg95. The C-terminal sequences of cruciferin 1 and vicilin-like storage protein 22 were sufficient to redirect the secretory red fluorescent protein (spRFP) to the vacuoles in Arabidopsis protoplasts. Adding a proline residue to the C terminus of the ctVSD and R95M substitution of VSR1 disrupted receptor–cargo interactions in vitro and led to increased secretion of spRFP in Arabidopsis protoplasts. How VSR1-PA recognizes ctVSDs of other storage proteins was modeled. The last three residues of ctVSD prefer hydrophobic residues because they form a hydrophobic cluster with Tyr99 of VSR1-PA. Due to charge–charge interactions, conserved acidic residues, Asp129 and Glu132, around the cargo-binding site should prefer basic residues over acidic ones in the ctVSD. The structural insights gained may be useful in targeting recombinant proteins to the protein storage vacuoles in seeds.

During seed development, storage proteins are deposited in a specialized organelle called the protein storage vacuole (PSV) and are mobilized to provide sources of carbon, nitrogen, and sulfur during germination (1). Seed storage proteins are synthesized as secretory proteins that are translocated into the endoplasmic reticulum (ER). How these proteins are transported to the PSV is not fully understood. In the receptor-mediated sorting pathway, storage proteins are sorted to the vacuoles via sequence-specific interactions with transmembrane sorting receptors (2). According to the latest model, sorting receptors could pick up the cargo proteins as early as in the ER and transport them through the Golgi apparatus to the trans-Golgi network (TGN), which then matures into the prevacuolar compartment (PVC) and PSV (2). Alternatively, storage proteins are concentrated and aggregated at the periphery of the cis-Golgi, where the dense vesicles (DVs) are formed (3, 4). DVs later bud off and fuse with the PVC, which matures into the PSV. Receptor–cargo interaction could play a role in the aggregation of storage proteins. For example, removal of the C-terminal hydrophobic (AFVY) residues of phaseolin, a storage protein of French bean (Phaseolus vulgaris), abolished aggregation of phaseolin and missorted it to the extracellular space (5).There are two families of sorting receptors, namely vacuolar sorting receptors (VSRs) and receptor-homology-transmembrane-RING-H2 (RMR) proteins (6–8). There are seven homologs of VSR and six homologs of RMR in the Arabidopsis thaliana genome. Unlike lysosomal sorting in animal cells that recognizes the posttranslational modification of mannose-6-phosphate (9), vacuolar sorting in yeast and plant cells is mediated by specific protein–protein interactions between the sorting receptors and the cargo proteins (6, 10–19). These sorting receptors recognize sequence-specific information, or vacuolar sorting determinants (VSDs), on the cargo proteins (20, 21). There are two types of VSD, the sequence-specific VSD (ssVSD) and the C-terminal VSD (ctVSD) (22–24). VSRs can recognize both ssVSDs and ctVSDs (6, 15, 25), while RMRs can only recognize ctVSDs (26, 27). ssVSD, often found in acidic hydrolases targeting lytic vacuoles, contains an NPIR motif with the consensus sequence of (N/L)-(P/I/L)-(I/P)-(R/N/S) (28). Mutations in the NPIR motif disrupt receptor–cargo interactions and lead to missorting of cargo proteins (18, 21, 29, 30). Unlike ssVSD that is located at internal sequence positions, ctVSD is only found at the C terminus of cargo proteins. No consensus sequence has been identified for ctVSD, but it is usually rich in hydrophobic residues (20). For example, the AFVY motif at the C terminus of phaseolin was found to be essential for targeting seed proteins to the PSV (31).VSRs are type I transmembrane proteins that contain a protease-associated (PA) domain, a central domain, and three epidermal growth factor (EGF) repeats in the luminal N-terminal region, followed by a single transmembrane domain (TMD) and a C-terminal cytoplasmic tail (Fig. 1A) (6, 12, 32). The PA domain and central domain are involved in sequence-specific interactions with ssVSDs (21, 33). We have previously determined the crystal structure of the PA domain of vacuolar sorting receptor isoform 1 (VSR1-PA) in complex with the ssVSD of barley aleurain (21) and showed that the PA domain is responsible for recognizing the sequences preceding the NPIR motif. Cargo binding induces the C-terminal tail to undergo a swivel motion that could relocate the central domain to cooperate with the PA domain for ssVSD recognition (21). The EGF repeats have unclear functions, but they might regulate the cargo binding by calcium-dependent conformational change of the PA and central domains (33, 34).Open in a separate windowFig. 1.Crystal structure of VSR1-PA in complex with the C-terminal pentapeptide (₄₆₈RVAAA₄₇₂) of CRU1. (A) Domain organization of VSRs. VSR1-NT consists of a protease-associated domain, a central domain, and three EGF repeats. sp, signal peptide. (B) Pull-down assay. E. coli–expressed VSR1-PA was incubated with NHS-resins coupled with the C-terminal peptide sequence of CRU1 (YRVAAA) or with glycine. A tyrosine residue was added to the N terminus of the peptide to facilitate the quantification of peptide concentration using A₂₈₀. After extensive washing to ensure VSR1-PA was not present in the last wash fractions (W), VSR1-PA bound (B) to the resins was analyzed by immunoblot with a VSR1-PA antibody. (C) Cartoon representation of the crystal structure of VSR1-PA in complex with the CRU1 C-terminal sequence, ₄₆₈RVAAA₄₇₂ (yellow). Switch I, II, and III regions and the cargo-binding loop are color-coded green, magenta, salmon, and cyan, respectively. The complex structure determined at pH 6.5 is shown. (D) A close-up view of the detailed receptor–cargo interactions. ₄₆₈RVA₄₇₀ of CRU1 forms a parallel β-sheet with ₁₂₈TMD₁₂₉ of switch III. The last two residues of CRU1, ₄₇₁AA₄₇₂, dock into a cradle formed by the conserved residues in the cargo-binding loop, ₉₅RGDCYF₁₀₀. The backbone conformation of the bound cargo is maintained by a number of backbone–backbone hydrogen bonds (dotted lines). The C-terminal carboxyl group of CRU1 forms salt bridges with Arg95 in the cargo-binding loop. Intermolecular hydrogen bonds and salt bridges are summarized (Right). (E) VSR1-PA undergoes conformational changes upon binding of ₄₆₈RVAAA₄₇₂. In the apo form of VSR1-PA (light blue), the cargo-binding site is occupied by switch III residues, where Glu133 forms salt bridges with Arg95. Cargo binding displaces the switch III residues away. N-terminal residues 20 to 24 that form strand β-1N in the apo form became disordered, making room for Asn46 in the switch II region to move toward and make a hydrogen bond with Met128 of switch III. Switch I residues (25 to 27) straighten up to form an antiparallel β-sheet with β-2. (F) Sequence of VSR1-PA, pumpkin PV72, pea BP-80, and soybean and French bean VSRs were aligned using the program MUSCLE (57). Secondary structure elements of the bound and apo forms of VSR1-PA are indicated above and below the alignment, respectively. Dotted lines indicate residues that are disordered in the crystal structures. Residues are numbered according to the VSR1-PA sequence.The role of VSRs in sorting seed storage proteins has been supported by genetic studies in Arabidopsis. In a pioneer study, Shimada and coworkers showed that the vsr1 knockout mutant missorted the seed storage proteins 12S globulin and 2S albumin to the extracellular space in Arabidopsis seeds (15). Zouhar and coworkers further showed that vsr1vsr3 and vsr1vsr4 double mutants reduced the amount of the mature form of 12S globulin in the PSV, suggesting that VSR1, VSR3, and VSR4 are the sorting receptors for 12S globulin (35). Moreover, tagging the C-terminal 24 residues of β-conglycinin to the C terminus of a secretory green fluorescent protein (GFP) was sufficient to target the fluorescent protein to the PSV in Arabidopsis seeds, while the fluorescent protein was missorted to the extracellular space in the vsr1 mutant (19). Since VSR1 can bind to the C-terminal sequence of both cruciferin 1 (CRU1), an isoform of 12S globulin, and β-conglycinin (15, 19), it is likely that VSR1 recognizes the sorting determinants in these sequences and sorts them to the PSV in seeds.The molecular mechanism of how VSRs recognize the sorting determinants of cargo proteins remains elusive. In this study, we report the crystal structure of the PA domain of VSR1 in complex with the C-terminal pentapeptide (₄₆₈RVAAA₄₇₂) of CRU1. Structural insights into receptor–cargo interaction were supported by mutagenesis and functional studies, which showed that a specific recognition between the VSR and ctVSD is essential for vacuolar sorting.     

Arf6 coordinates actin assembly through the WAVE complex,a mechanism usurped by Salmonella to invade host cells

ADP ribosylation factor (Arf) 6 anchors to the plasma membrane, where it coordinates membrane trafficking and cytoskeleton remodelling, but how it assembles actin filaments is unknown. By reconstituting membrane-associated actin assembly mediated by the WASP family veroprolin homolog (WAVE) regulatory complex (WRC), we recapitulated an Arf6-driven actin polymerization pathway. We show that Arf6 is divergent from other Arf members, as it was incapable of directly recruiting WRC. We demonstrate that Arf6 triggers actin assembly at the membrane indirectly by recruiting the Arf guanine nucleotide exchange factor (GEF) ARNO that activates Arf1 to enable WRC-dependent actin assembly. The pathogen Salmonella usurped Arf6 for host cell invasion by recruiting its canonical GEFs EFA6 and BRAG2. Arf6 and its GEFs facilitated membrane ruffling and pathogen invasion via ARNO, and triggered actin assembly by generating an Arf1–WRC signaling hub at the membrane in vitro and in cells. This study reconstitutes Arf6-dependent actin assembly to reveal a mechanism by which related Arf GTPases orchestrate distinct steps in the WRC cytoskeleton remodelling pathway.ADP ribosylation factor (Arf) GTPases are best known for their roles in vesicle and organelle trafficking (1). Class I and II Arfs (Arf1, Arf3, Arf4, and Arf5) are found predominantly in and around the Golgi apparatus. In contrast, the more divergent Class III Arf (Arf6) operates almost exclusively at the plasma membrane (2). Consistent with its localization, Arf6 has been heavily implicated in trafficking events at the cell surface, including the regulation of endocytosis and exocytosis (1). In particular, Arf6 and its guanine nucleotide exchange factors (GEFs) are believed to be pivotal to the recycling of endosomes and receptors to and from the plasma membrane (3, 4). Arf6 also has a clear role in cortical cytoskeleton rearrangement (5). This is strongly supported by evidence of Arf6 and Rac1 (Ras-related C3 botulinum toxin substrate) interplay (6), exemplified by Arf6 recruitment of the Rac1 GEF Kalirin, Arf6 promotion of Rac1 activation, and lamellipodia formation (7–9).Rac1 is required for generation of lamellipodia that lead to membrane ruffles and macropinocytosis (10). Rac1 is thought to achieve this by activating the WRC, which comprises WAVE (WASP family veroprolin homolog), Abi (abl-interactor 1), Cyfip (cytoplasmic FMR1 interacting protein), Nap1 (NCK-associated protein 1), HSPC300 (heat shock protein C300), or their homologs (10–12). We recently established that Rac1 was not sufficient for WRC recruitment to the membrane and its activation, which instead requires direct binding by Rac1 and an Arf GTPase (13). This in vitro Arf activity could be supplied by multiple isoforms including Arf1 and Arf5, but only Arf1 facilitated WRC-dependent lamellipodia formation and macropinocytosis of the bacterial pathogen Salmonella into human host cells (13–15). Intriguingly, Arf6 also promoted Salmonella invasion (14), and, given the capability of Arfs to modulate WRC, it seems likely that Arf6 also recruits and activates the WRC at the plasma membrane. However, despite mounting evidence that Arf6 remodels the cytoskeleton, a molecular mechanism by which Arf6 drives Arp2/3-dependent actin assembly has not been resolved.     

Structural basis for membrane recruitment and allosteric activation of cytohesin family Arf GTPase exchange factors

Membrane recruitment of cytohesin family Arf guanine nucleotide exchange factors depends on interactions with phosphoinositides and active Arf GTPases that, in turn, relieve autoinhibition of the catalytic Sec7 domain through an unknown structural mechanism. Here, we show that Arf6-GTP relieves autoinhibition by binding to an allosteric site that includes the autoinhibitory elements in addition to the PH domain. The crystal structure of a cytohesin-3 construct encompassing the allosteric site in complex with the head group of phosphatidyl inositol 3,4,5-trisphosphate and N-terminally truncated Arf6-GTP reveals a large conformational rearrangement, whereby autoinhibition can be relieved by competitive sequestration of the autoinhibitory elements in grooves at the Arf6/PH domain interface. Disposition of the known membrane targeting determinants on a common surface is compatible with multivalent membrane docking and subsequent activation of Arf substrates, suggesting a plausible model through which membrane recruitment and allosteric activation could be structurally integrated.Guanine nucleotide exchange factors (GEFs) activate GTPases by catalyzing exchange of GDP for GTP (1). Because many GEFs are recruited to membranes through interactions with phospholipids, active GTPases, or other membrane-associated proteins (1–5), GTPase activation can be restricted or amplified by spatial–temporal overlap of GEFs with binding partners. GEF activity can also be controlled by autoregulatory mechanisms, which may depend on membrane recruitment (6–11). Structural relationships between these mechanisms are poorly understood.Arf GTPases function in trafficking and cytoskeletal dynamics (5, 12, 13). Membrane partitioning of a myristoylated (myr) N-terminal amphipathic helix primes Arfs for activation by Sec7 domain GEFs (14–17). Cytohesins comprise a metazoan Arf GEF family that includes the mammalian proteins cytohesin-1 (Cyth1), ARNO (Cyth2), and Grp1 (Cyth3). The Drosophila homolog steppke functions in insulin-like growth factor signaling, whereas Cyth1 and Grp1 have been implicated in insulin signaling and Glut4 trafficking, respectively (18–20). Cytohesins share a modular architecture consisting of heptad repeats, a Sec7 domain with exchange activity for Arf1 and Arf6, a PH domain that binds phosphatidyl inositol (PI) polyphosphates, and a C-terminal helix (CtH) that overlaps with a polybasic region (PBR) (21–28). The overlapping CtH and PBR will be referred to as the CtH/PBR. The phosphoinositide specificity of the PH domain is influenced by alternative splicing, which generates diglycine (2G) and triglycine (3G) variants differing by insertion of a glycine residue in the β1/β2 loop (29). Despite similar PI(4,5)P₂ (PIP₂) affinities, the 2G variant has 30-fold higher affinity for PI(3,4,5)P₃ (PIP₃) (30). In both cases, PIP₃ is required for plasma membrane (PM) recruitment (23, 26, 31–33), which is promoted by expression of constitutively active Arf6 or Arl4d and impaired by PH domain mutations that disrupt PIP₃ or Arf6 binding, or by CtH/PBR mutations (8, 34–36).Cytohesins are autoinhibited by the Sec7-PH linker and CtH/PBR, which obstruct substrate binding (8). Autoinhibition can be relieved by Arf6-GTP binding in the presence of the PIP₃ head group (8). Active myr-Arf1 and myr-Arf6 also stimulate exchange activity on PIP₂-containing liposomes (37). Whether this effect is due to relief of autoinhibition per se or enhanced membrane recruitment is not yet clear. Phosphoinositide recognition by PH domains, catalysis of nucleotide exchange by Sec7 domains, and autoinhibition in cytohesins are well characterized (8, 16, 17, 30, 38–43). How Arf-GTP binding relieves autoinhibition and promotes membrane recruitment is unknown. Here, we determine the structural basis for relief of autoinhibition and investigate potential mechanistic relationships between allosteric regulation, phosphoinositide binding, and membrane targeting.     

Lysine 63-linked polyubiquitination is required for EGF receptor degradation

Ubiquitination mediates endocytosis and endosomal sorting of various signaling receptors, transporters, and channels. However, the relative importance of mono- versus polyubiquitination and the role of specific types of polyubiquitin linkages in endocytic trafficking remain controversial. We used mass spectrometry-based targeted proteomics to show that activated epidermal growth factor receptor (EGFR) is ubiquitinated by one to two short (two to three ubiquitins) polyubiquitin chains mainly linked via lysine 63 (K63) or conjugated with a single monoubiquitin. Multimonoubiquitinated EGFR species were not found. To directly test whether K63 polyubiquitination is necessary for endocytosis and post-endocytic sorting of EGFR, a chimeric protein, in which the K63 linkage-specific deubiquitination enzyme AMSH [associated molecule with the Src homology 3 domain of signal transducing adaptor molecule (STAM)] was fused to the carboxyl terminus of EGFR, was generated. MS analysis of EGFR-AMSH ubiquitination demonstrated that the fraction of K63 linkages was substantially reduced, whereas relative amounts of monoubiquitin and K48 linkages increased, compared with that of wild-type EGFR. EGFR-AMSH was efficiently internalized into early endosomes, but, importantly, the rates of ligand-induced sorting to late endosomes and degradation of EGFR-AMSH were dramatically decreased. The slow degradation of EGFR-AMSH resulted in the sustained signaling activity of this chimeric receptor. Ubiquitination patterns, rate of endosomal sorting, and signaling kinetics of EGFR fused with the catalytically inactive mutant of AMSH were reversed to normal. Altogether, the data are consistent with the model whereby short K63-linked polyubiquitin chains but not multimonoubiquitin provide an increased avidity for EGFR interactions with ubiquitin adaptors, thus allowing rapid sorting of activated EGFR to the lysosomal degradation pathway.Ubiquitination, a posttranslational modification of proteins by attachment of the ubiquitin (Ub) polypeptide, is an important molecular signal that regulates endocytosis and post-endocytic sorting of membrane proteins (1–3). Ubiquitination is carried out by the sequential activity of E1, E2, and E3 enzymes; the latter, E3 ligases, typically determine the substrate specificity of Ub conjugation (4). Deubiquitinating enzymes (DUBs), a group of proteases capable of cleaving Ub from conjugates with target proteins, counteract the activity of the ubiquitination system (5). Ub is predominantly conjugated to lysine residues and much more rarely to the amino-terminal methionine or other amino acids in the substrate. Lysines and the amino-terminal methionine in the Ub molecule can also be conjugated to another molecule of Ub, leading to the formation of polyUb chains (6). Depending on the specific residue that links Ubs into a chain, polyUb chains have different molecular folding, are recognized by specific Ub-binding domains (UBDs) and have distinct functions (7). The structure and interaction mechanisms of lysine 48 (K48)- and K63-linked chains are most well-characterized (8–12). Crystal and NMR structures of K63 di-Ubs revealed extended, open conformation of two Ubs with high conformational freedom, as opposed to closed conformation of K48-polyUb linkages (reviewed in ref. 11). Therefore, ubiquitination substrates including endocytic cargo can be mono- and polyubiquitinated by different chains, but the role of these diverse types of ubiquitination in the regulation of endocytic trafficking remains incompletely understood.Epidermal growth factor (EGF) receptor (EGFR) was one of the first endocytic cargos in mammalian cells that were found to be ubiquitinated (13). This receptor has the profound role in eukaryotic development, regulation of various tissues in adult organisms, and pathogenesis of cancer (14). Therefore, EGFR has been a prototypic model for studying the mechanisms of endocytosis and endocytosis-relevant ubiquitination. EGFR is ubiquitinated by Cbl E3 ligases at the cell surface and after internalization in endosomes (15–17). The internalization step of EGFR trafficking is regulated by multiple redundant mechanisms, including ubiquitination, and is not significantly inhibited in the absence of receptor ubiquitination (18). By contrast, sorting of the internalized receptor in multivesicular bodies (MVBs), which leads to its incorporation into intraluminal vesicles of MVB and degradation in lysosomes, is highly sensitive to the extent of EGFR ubiquitination (15, 19).Based on differential recognition by Ub antibodies, EGFR was proposed to be conjugated with multiple monoUbs (20). Moreover, replacement of the cytoplasmic domain of EGFR with the Ub mutant incapable of polyubiquitination resulted in EGF-independent endocytosis and degradation of such chimeric receptor, thus suggesting that monoubiquitination is sufficient for EGFR endocytosis and MVB sorting (20). Subsequently, mass spectrometric (MS) analysis demonstrated a significant amount of EGFR polyubiquitination, mainly by K63-linked chains (19, 21, 22). However, whether K63 polyubiquitination is necessary for EGFR endocytic trafficking remains unknown.The role of cargo ubiquitination by K63-linked chains has been proposed in studies of endocytosis and MVB sorting of yeast permeases (23–27). These studies, however, used an approach of global elimination of K63 polyubiquitination in the cell to demonstrate the importance of these chains in endocytic trafficking. Because numerous proteins, including ESCRT components mediating MVB sorting are polyubiquitinated with K63 linkages, the inhibitory effects of the blockade of K63-linked polyubiquitination on endocytosis and MVB sorting observed in these studies may be indirect [e.g., not related to cargo ubiquitination (25)]. By contrast, an alternative approach based on the analysis of genetically engineered chimeric cargo molecules fused to Ub or a DUB demonstrated that monoubiquitination is fully sufficient for endocytosis and sorting of several membrane proteins to the vacuole in yeast (28).A number of mammalian endocytic cargo is polyubiquitinated by K63-linked chains (29–31) and, to a lesser extent, with K48 linkages (32–35). Similarly to studies in yeast, the role of these Ub linkages in mammalian cells was mainly examined by overexpressing K63R or K48R Ub mutants incapable of forming corresponding polyUb chains, leading to inhibition of K63 or K48 polyubiquitination of all cellular substrates (31–33). To test whether K63 polyubiquitination is required for EGFR endocytosis and endosomal sorting, we analyzed the stoichiometry of EGFR ubiquitination by MS and generated a chimeric EGFR fused at the carboxyl terminus to a DUB with the specificity toward K63 linkages. Analysis of the internalization and post-endocytic sorting of this chimeric receptor showed that K63 polyUb chains are necessary for the efficient EGF-induced down-regulation of EGFR.     

Overlapping functions of stonin 2 and SV2 in sorting of the calcium sensor synaptotagmin 1 to synaptic vesicles

Neurotransmission involves the calcium-regulated exocytic fusion of synaptic vesicles (SVs) and the subsequent retrieval of SV membranes followed by reformation of properly sized and shaped SVs. An unresolved question is whether each SV protein is sorted by its own dedicated adaptor or whether sorting is facilitated by association between different SV proteins. We demonstrate that endocytic sorting of the calcium sensor synaptotagmin 1 (Syt1) is mediated by the overlapping activities of the Syt1-associated SV glycoprotein SV2A/B and the endocytic Syt1-adaptor stonin 2 (Stn2). Deletion or knockdown of either SV2A/B or Stn2 results in partial Syt1 loss and missorting of Syt1 to the neuronal surface, whereas deletion of both SV2A/B and Stn2 dramatically exacerbates this phenotype. Selective missorting and degradation of Syt1 in the absence of SV2A/B and Stn2 impairs the efficacy of neurotransmission at hippocampal synapses. These results indicate that endocytic sorting of Syt1 to SVs is mediated by the overlapping activities of SV2A/B and Stn2 and favor a model according to which SV protein sorting is guarded by both cargo-specific mechanisms as well as association between SV proteins.Neurotransmission is based on the calcium-triggered fusion of neurotransmitter-filled synaptic vesicles (SVs) with the presynaptic plasma membrane. To sustain neurotransmitter release, neurons have evolved mechanisms to retrieve SV membranes and to reform SVs locally within presynaptic nerve terminals. How SVs are reformed and maintain their compositional identity (1, 2) is controversial (3–5). One possibility is that upon fusion SV proteins remain clustered at the active zone—that is, by association between SV proteins—and are retrieved via “kiss-and-run” or ultrafast endocytosis (6), thereby alleviating the need for specific sorting of individual SV proteins. Alternatively, if SVs lose their identity during multiple rounds of exo-/endocytosis (7, 8), specific mechanisms exist to orchestrate high-fidelity SV protein sorting, either directly at the plasma membrane via slow clathrin-mediated endocytosis (CME) or at endosome-like vacuoles generated by fast clathrin-independent membrane retrieval (5, 9). Endocytic adaptors for SV protein sorting include the heterotetrameric adaptor protein complex 2 (AP-2) (9), the synaptobrevin 2/VAMP2 adaptor AP180 (10), and the AP-2μ–related protein stonin 2 (Stn2), a specific sorting adaptor for the SV calcium sensor synaptotagmin 1 (Syt1) (8, 11). Although genetic inactivation of the Stn2 orthologs Stoned B and Unc41 in flies and worms is lethal due to defective neurotransmission caused by degradation and missorting of Syt1 (12, 13), Stn2 knockout (KO) mice are viable and able to internalize Syt1, albeit with reduced fidelity of sorting (14). Thus, mammalian synapses, in contrast to invertebrates, have evolved mechanisms to sort Syt1 in the absence of its specific sorting adaptor Stn2. One possibility is that Syt1 sorting in addition to its direct recognition by Stn2 is facilitated by complex formation with other SV proteins. Likely candidates for such a piggyback mechanism are the SV2 family of transmembrane SV glycoproteins (15, 16), which might regulate Syt1 function either via direct interaction (17, 18) or by facilitating its binding to AP-2 (19). Apart from the distantly related SVOP protein (20), no close SV2 homologs exist in invertebrates, suggesting that SV2 fulfills a unique function at mammalian synapses. KO of SV2A or combined loss of its major A and B isoforms in mice causes early postnatal lethality due to epileptic seizures (21, 22), impaired neurotransmission (23, 24), and defects in Syt1 trafficking (25), whereas SV2B KO mice are phenotypically normal (17). Given that SV2A in addition to its association with Syt1 binds to endocytic proteins including AP-2 and Eps15 (25), SV2 would be a likely candidate for mediating Syt1 sorting to SVs.Here we demonstrate that endocytic sorting of Syt1 is mediated by the overlapping activities of SV2A/B and Stn2. Deletion or knockdown of either SV2A/B or Stn2 results in partial Syt1 loss and missorting of Syt1 to the neuronal surface, whereas deletion of both SV2A/B and Stn2 dramatically exacerbates this phenotype, resulting in severely impaired basal neurotransmission. Our results favor a model according to which SV protein sorting is guarded by both cargo-specific mechanisms as well as association between SV proteins.     

PKD-dependent PARP12-catalyzed mono-ADP-ribosylation of Golgin-97 is required for E-cadherin transport from Golgi to plasma membrane

Adenosine diphosphate (ADP)-ribosylation is a posttranslational modification involved in key regulatory events catalyzed by ADP-ribosyltransferases (ARTs). Substrate identification and localization of the mono-ADP-ribosyltransferase PARP12 at the trans-Golgi network (TGN) hinted at the involvement of ARTs in intracellular traffic. We find that Golgin-97, a TGN protein required for the formation and transport of a specific class of basolateral cargoes (e.g., E-cadherin and vesicular stomatitis virus G protein [VSVG]), is a PARP12 substrate. PARP12 targets an acidic cluster in the Golgin-97 coiled-coil domain essential for function. Its mutation or PARP12 depletion, delays E-cadherin and VSVG export and leads to a defect in carrier fission, hence in transport, with consequent accumulation of cargoes in a trans-Golgi/Rab11–positive intermediate compartment. In contrast, PARP12 does not control the Golgin-245–dependent traffic of cargoes such as tumor necrosis factor alpha (TNFα). Thus, the transport of different basolateral proteins to the plasma membrane is differentially regulated by Golgin-97 mono-ADP-ribosylation by PARP12. This identifies a selective regulatory mechanism acting on the transport of Golgin-97– vs. Golgin-245–dependent cargoes. Of note, PARP12 enzymatic activity, and consequently Golgin-97 mono-ADP-ribosylation, depends on the activation of protein kinase D (PKD) at the TGN during traffic. PARP12 is directly phosphorylated by PKD, and this is essential to stimulate PARP12 catalytic activity. PARP12 is therefore a component of the PKD-driven regulatory cascade that selectively controls a major branch of the basolateral transport pathway. We propose that through this mechanism, PARP12 contributes to the maintenance of E-cadherin–mediated cell polarity and cell–cell junctions.

Adenosine diphosphate (ADP) ribosylation is a protein posttranslational modification (PTM) consisting of the transfer of an ADP-ribose moiety from NAD⁺ to target amino acids that is highly conserved throughout evolution (1–3). The enzymes catalyzing this reaction, named ADP-ribosyltransferases (ARTs), first diversified in bacteria into a variety of systems involved in defensive and offensive strategies in intragenomic, intergenomic, and intraorganismal conflicts, and have been acquired by eukaryotes from these conflict systems several times throughout evolution (1, 4). In eukaryotes, ADP-ribosyltransferases are often components of core regulatory and epigenetic processes (5–7). The analysis of their eukaryotic substrates is thus likely to provide information on the organization and regulation of key cellular functions.The ARTs (8) constitute a major family of ADP-ribosyltransferases whose members catalyze ADP-ribosylation by adding either single or multiple units of the NAD⁺-deriving ADP-ribose onto target proteins [respectively, mono- and poly-ADP-ribosylation, hereafter referred to as MARylation and PARylation (9)]. MARylation of mammalian proteins was first discovered decades ago to mediate the pathogenic action of bacterial toxins in host cells (10, 11). The endogenous occurrence of this PTM in mammalian cells later became evident (11–16) and, recently, with the definition of the different enzymes catalyzing the reaction, the cellular functions it regulates are emerging (17–19).So far, eukaryotic ADP-ribosylation has been mainly studied under stress conditions, as exemplified by the role of poly (ADP-ribose) polymerase 1 (PARP1)–mediated PARylation during the DNA-damage response (20), PARP5, -12, and -13 in stress-granule formation (21–23), or PARP16 in the unfolded protein response (24, 25), while its impact on physiological cellular processes remains poorly defined.Intracellular membrane transport is emerging as a function regulated by PARPs, with particular reference to Golgi-localized PARPs, namely PARP5 and -12 (26). PARP5 (also called tankyrase) is known to regulate the delivery of the glucose transporter GLUT4 from the trans-Golgi network (TGN) to glucose-storage vesicles and thus to the plasma membrane [PM (27–30)]. PARP12, originally described to be involved in defense against viral infections (31–34), is involved in the anterograde transport of the vesicular stomatitis virus G protein (VSVG) from the TGN to the PM (21, 26, 35) and is a well-known component of stress granules, where it translocates from the Golgi upon oxidative stress (21, 23).The TGN is a major sorting station where cargoes are conveyed and sorted into distinct transport carriers for trafficking to post-Golgi compartments and to the PM (36). The different trafficking routes undertaken by individual cargoes are regulated by transport machineries, including small G proteins belonging to the ADP-ribosylation factor (Arf) and Rab families, cytosolic cargo-adaptor proteins, coat proteins, and accessory proteins, all involved in cargo “packaging” into specific transport carriers to achieve correct sorting and delivery (36–38).Here, we report that PARP12 controls the basolateral transport of a subclass of basolateral cargoes, which includes VSVG and E-cadherin, through the MARylation of Golgin-97. Moreover, we find that PARP12-mediated MARylation requires the presence of protein kinase D (PKD), a master regulator of basolateral transport (39, 40), and that it is stimulated by PKD during cargo trafficking. Traffic-activated PKD phosphorylates PARP12, activating its enzymatic activity. It thus emerged that PARP12-mediated MARylation of Golgin-97 is a component of the PKD-dependent regulatory network underlying the basolateral secretion of a select subgroup of cargo proteins, including E-cadherin. Since E-cadherin is required for the formation of proper adherens junctions and epithelial polarization, we propose that the regulatory cascade described in this study may play a role in the maintenance of cellular polarity in epithelial cells and therefore in various body functions (41, 42).     

Calcium levels in the Golgi complex regulate clustering and apical sorting of GPI-APs in polarized epithelial cells

Glycosylphosphatidylinositol-anchored proteins (GPI-APs) are lipid-associated luminal secretory cargoes selectively sorted to the apical surface of the epithelia where they reside and play diverse vital functions. Cholesterol-dependent clustering of GPI-APs in the Golgi is the key step driving their apical sorting and their further plasma membrane organization and activity; however, the specific machinery involved in this Golgi event is still poorly understood. In this study, we show that the formation of GPI-AP homoclusters (made of single GPI-AP species) in the Golgi relies directly on the levels of calcium within cisternae. We further demonstrate that the TGN calcium/manganese pump, SPCA1, which regulates the calcium concentration within the Golgi, and Cab45, a calcium-binding luminal Golgi resident protein, are essential for the formation of GPI-AP homoclusters in the Golgi and for their subsequent apical sorting. Down-regulation of SPCA1 or Cab45 in polarized epithelial cells impairs the oligomerization of GPI-APs in the Golgi complex and leads to their missorting to the basolateral surface. Overall, our data reveal an unexpected role for calcium in the mechanism of GPI-AP apical sorting in polarized epithelial cells and identify the molecular machinery involved in the clustering of GPI-APs in the Golgi.

Glycosylphosphatidylinositol (GPI)-anchored proteins (GPI-APs) are localized on the apical surface of most epithelia, where they exert their physiological functions, which are regulated by their spatiotemporal compartmentalization.In polarized epithelial cells, the organization of GPI-APs at the apical surface is driven by the mechanism of apical sorting, which relies on the formation of GPI-AP homoclusters in the Golgi apparatus (1, 2). GPI-AP homoclusters (containing a single GPI-AP species) form uniquely in the Golgi apparatus of fully polarized cells (and not in nonpolarized cells) in a cholesterol-dependent manner (1, 3, 4). Once formed, GPI-AP homoclusters become insensitive to cholesterol depletion, suggesting that protein–protein interactions stabilize them (1, 2). At the apical membrane, newly arrived homoclusters coalesce into heteroclusters (containing at least two different GPI-AP species) that are sensitive to cholesterol depletion (1). Of importance, in the absence of homoclustering in the Golgi (e.g., in nonpolarized epithelial cells), GPI-APs remain in the form of monomers and dimers and do not cluster at the cell surface (1, 5). Thus, the organization of GPI-APs at the apical plasma membrane of polarized cells strictly depends on clustering mechanisms in the Golgi apparatus allowing their apical sorting. This is different from what was shown in fibroblasts where clustering of GPI-APs occurs from monomer condensation at the plasma membrane, indicating that distinct mechanisms regulate GPI-AP clustering in polarized epithelial cells and fibroblasts (1, 6, 7). Furthermore, in polarized epithelial cells, the spatial organization of clusters also appears to regulate the biological activity of the proteins (1) so that GPI-APs are fully functional only when properly sorted to the apical surface and less active in the case of missorting to the basolateral domain (1, 8, 9). Understanding the mechanism of GPI-AP apical sorting in the Golgi apparatus is therefore crucial to decipher their organization at the plasma membrane and the regulation of their activity. The determinants for protein apical sorting have been difficult to uncover compared to the ones for basolateral sorting (10–14). Besides a role of cholesterol, the molecular factors regulating the clustering-based mechanism of GPI-AP sorting in polarized epithelial cells are unknown. Here, we analyzed the possible role of the actin cytoskeleton and of calcium levels in the Golgi. The actin cytoskeleton is not only critical for the maintenance of the Golgi structure and its mechanical properties but also provides the structural support favoring carrier biogenesis (15–18). The Golgi exit of various cargoes is altered in cells treated with drugs either depolymerizing or stabilizing actin filaments (19, 20), and the post-Golgi trafficking is affected either by the knockdown of the expression of some actin-binding proteins, which regulate actin dynamics, or by the overexpression of their mutants (12, 21–23), all together revealing the critical role of actin dynamics for protein trafficking. Only few studies have shown the involvement of actin remodeling proteins in polarized trafficking, mostly in selectively mediating the apical and basolateral trafficking of transmembrane proteins [refs. 24–26; and reviewed in ref. 27]; thus, it remains unclear whether actin filaments play a role in protein sorting in polarized cells.On the other hand, the Golgi apparatus exhibits high calcium levels that have been revealed to be essential for protein processing and the sorting of some secreted soluble proteins in nonpolarized cells (28–31). Moreover, a functional interplay between the actin cytoskeleton and Golgi calcium in modulating protein sorting in nonpolarized cells has been shown (22).In this study, we report that in epithelial cells, actin perturbation does not impair GPI-AP clustering capacity in the Golgi and therefore their apical sorting. In contrast, we found that the Golgi organization of GPI-APs is drastically perturbed upon calcium depletion and that the amount of calcium in the Golgi cisternae is critical for the formation of GPI-AP homoclusters. We further show that the TGN calcium/manganese pump, SPCA1 (secretory pathway Ca(2+)-ATPase pump type 1), which controls the Golgi calcium concentration (32), and Cab45, a calcium-binding luminal Golgi resident protein previously described to be involved in the sorting of a subset of soluble cargoes (33, 34), are essential for the formation of GPI-APs homoclusters in the Golgi and for their subsequent apical sorting. Indeed, down-regulation of SPCA1 or Cab45 expression impairs the oligomerization of GPI-APs in the Golgi complex and leads to their missorting to the basolateral surface but does not affect apical or basolateral transmembrane proteins. Overall, our data reveal an unexpected role for calcium in the mechanism of GPI-AP apical sorting in polarized epithelial cells and identify the molecular machinery involved in the clustering of GPI-APs in the Golgi.     

EFA6 controls Arf1 and Arf6 activation through a negative feedback loop

Guanine nucleotide exchange factors (GEFs) of the exchange factor for Arf6 (EFA6), brefeldin A-resistant Arf guanine nucleotide exchange factor (BRAG), and cytohesin subfamilies activate small GTPases of the Arf family in endocytic events. These ArfGEFs carry a pleckstrin homology (PH) domain in tandem with their catalytic Sec7 domain, which is autoinhibitory and supports a positive feedback loop in cytohesins but not in BRAGs, and has an as-yet unknown role in EFA6 regulation. In this study, we analyzed how EFA6A is regulated by its PH and C terminus (Ct) domains by reconstituting its GDP/GTP exchange activity on membranes. We found that EFA6 has a previously unappreciated high efficiency toward Arf1 on membranes and that, similar to BRAGs, its PH domain is not autoinhibitory and strongly potentiates nucleotide exchange on anionic liposomes. However, in striking contrast to both cytohesins and BRAGs, EFA6 is regulated by a negative feedback loop, which is mediated by an allosteric interaction of Arf6-GTP with the PH-Ct domain of EFA6 and monitors the activation of Arf1 and Arf6 differentially. These observations reveal that EFA6, BRAG, and cytohesins have unanticipated commonalities associated with divergent regulatory regimes. An important implication is that EFA6 and cytohesins may combine in a mixed negative-positive feedback loop. By allowing EFA6 to sustain a pool of dormant Arf6-GTP, such a circuit would fulfill the absolute requirement of cytohesins for activation by Arf-GTP before amplification of their GEF activity by their positive feedback loop.Guanine nucleotide exchange factors (GEFs), which activate small GTPases by stimulating their intrinsically very slow GDP/GTP exchange, are key players in the extraordinary diversity of small GTPases pathways (reviewed in ref. 1). Small GTPases carry little specificity determinants on their own to determine when and where they should be turned on and which pathway they should activate (2), which are instead largely monitored by their GEFs. Thus, understanding how different members of a GEF family activate an individual small GTPase in distinct patterns is a major issue in small GTPase biology in normal cells and in diseases.An important contribution to the functional specificity of GEFs is how they themselves are regulated. Crystallographic studies combined with biochemical studies that reconstituted GEF-stimulated GDP/GTP nucleotide exchange have been instrumental in uncovering a growing complexity of regulatory mechanisms (reviewed in ref. 1). These include autoinhibitory elements outside the catalytic GEF domain that block access to the active site (3–7), large conformational changes that release autoinhibition in response to various stimuli (8–11), positive feedback loops in which freshly produced GTP-bound GTPases stimulate GDP/GTP exchange (10, 12–15), and potentiation of nucleotide exchange by colocalization on membranes (11, 13, 16, 17).These previous studies demonstrated that a wide range of regulatory regimes can be achieved even at the scale of a single GEF family by regulatory mechanisms that combine in multiple ways. GEFs that activate small GTPases of the Arf family (ArfGEFs), which are major regulators of many aspects of membrane traffic and organelle structure in eukaryotic cells (reviewed in refs. 18 and 19), form one of the best-characterized GEF families to date (reviewed in ref. 1), making a comprehensive view of their regulatory repertoire within reach. ArfGEFs comprise two major groups: the BIG/GBF1 group, which functions at the Golgi, and a group composed of the exchange factor for Arf6 (EFA6), brefeldin A-resistant Arf guanine nucleotide exchange factor (BRAG), and cytohesin subfamilies, which activate Arf GTPases at the cell periphery and function in various aspects of endocytosis (reviewed in ref. 20). The actual substrates of these ArfGEFs have been difficult to establish, notably because the most abundant Arf isoform, Arf1, was long believed to be excluded from the plasma membrane where the Arf6 isoform is located. Accordingly, cytohesins and BRAGs have been described as Arf6-specific GEFs in cells but are now recognized as active Arf1-GEFs (16, 21, 22), whereas EFA6 remains the sole ArfGEF considered to be strictly Arf6-specific (23, 24).Members of the EFA6, BRAG, and cytohesin subfamilies have divergent N-terminal domains but a related domain organization in their C terminus comprising a Sec7 domain, which stimulates GDP/GTP exchange, followed by a pleckstrin homology (PH) domain, which has multiple regulatory functions. In cytohesins, the PH domain recognizes signaling phosphoinositides by its canonical lipid-binding site (25), autoinhibits the Sec7 domain by obstructing its Arf-binding site (4), and amplifies nucleotide exchange by a positive feedback loop involving its direct interaction with Arf1-GTP or Arf6-GTP (10, 13, 21). In contrast, the PH domain of BRAG is not autoinhibitory and is not involved in a feedback loop, but instead strongly potentiates nucleotide exchange by binding to polyanionic membranes without marked phosphoinositides preference (16).How members of the EFA6 subfamily are regulated is currently unknown. These ArfGEFs are found predominantly (although not exclusively) in the brain and function in the coordination of endocytosis and actin dynamics (23, 26, 27), in the maintenance of tight junctions (28), in microtubule dynamics in Caenorhabditis elegans embryos (29), and in the formation and maintenance of dendrites (30), although the molecular details of these functions remain largely unknown. Consistent with an important role in the brain, defects in EFA6 functions have been found in neurologic disorders (31) and in human gliomas (32). The PH domain of EFA6 subfamily members drives the localization of EFA6 members to the plasma membrane (26) and it binds to PIP₂ lipids (33). It is followed by a 150-residue C-terminal (Ct) domain predicted to form a coiled coil, which massively induces actin-rich membrane protrusions when expressed with the PH domain (26). The divergence of regulatory mechanisms between Sec7-PH–containing cytohesins and BRAGs prompted us to undertake a quantitative biochemical investigation of EFA6 nucleotide exchange regulation. Our findings reveal an overlooked dual specificity of EFA6 for Arf1 and Arf6 and an unprecedented regulation by a negative feedback loop, with important potential implications for the activation of Arf GTPases in endocytic events.     

Motor coupling through lipid membranes enhances transport velocities for ensembles of myosin Va

Myosin Va is an actin-based molecular motor responsible for transport and positioning of a wide array of intracellular cargoes. Although myosin Va motors have been well characterized at the single-molecule level, physiological transport is carried out by ensembles of motors. Studies that explore the behavior of ensembles of molecular motors have used nonphysiological cargoes such as DNA linkers or glass beads, which do not reproduce one key aspect of vesicular systems—the fluid intermotor coupling of biological lipid membranes. Using a system of defined synthetic lipid vesicles (100- to 650-nm diameter) composed of either 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) (fluid at room temperature) or 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) (gel at room temperature) with a range of surface densities of myosin Va motors (32–125 motors per μm²), we demonstrate that the velocity of vesicle transport by ensembles of myosin Va is sensitive to properties of the cargo. Gel-state DPPC vesicles bound with multiple motors travel at velocities equal to or less than vesicles with a single myosin Va (∼450 nm/s), whereas surprisingly, ensembles of myosin Va are able to transport fluid-state DOPC vesicles at velocities significantly faster (>700 nm/s) than a single motor. To explain these data, we developed a Monte Carlo simulation that suggests that these reductions in velocity can be attributed to two distinct mechanisms of intermotor interference (i.e., load-dependent modulation of stepping kinetics and binding-site exclusion), whereas faster transport velocities are consistent with a model wherein the normal stepping behavior of the myosin is supplemented by the preferential detachment of the trailing motor from the actin track.Myosin Va is a processive, actin-based molecular motor critical for transport, morphology, and positioning of a wide variety of intracellular cargoes and organelles (1, 2). Although a single myosin Va can transport cargo in vitro, intracellular membrane-bound vesicular cargoes have a high surface density of motor proteins (3–5). Ensembles of motors that transport cargo in vitro have been shown to demonstrate enhanced run lengths (6–9) and slower movement, relative to the behavior of a single motor (6, 8–13). Slower velocities are generally attributed to negative interference between motors that arise when rigidly coupled, high duty ratio motors step asynchronously (12). The stepping kinetics of myosin Va are load sensitive, whereby resistive loads slow the stepping rate, whereas assistive loads produce only a modest acceleration (14–16). This asymmetric load dependence causes an overall slowing for a motor ensemble. Indicative of the complexity of the intracellular environment, Efremov et al. (17) have shown that physiological vesicle transport (whether microtubule- or actin-based) does not directly mirror the behavior of the responsible molecular motor but is sensitive to aspects of the cargo itself. In some microtubule-based transport systems, velocities faster than the capacity of the transporting motor have been observed in cells (18, 19). However, motion of the cytoskeletal track may contribute to this enhanced motion (20).Most studies of in vitro cargo transport have a motor protein ensemble coupled to rigid cargoes such as quantum dots (Qdots) (8, 21), silica beads (12), DNA scaffolds (9, 22, 23), or directly to glass substrates (24). However, intracellular vesicles have fluid membranes that would be expected to allow the vesicle-attached motors (or coupled groups of motors) to diffuse within the membrane, so that motor ensemble transport of lipid-bound vesicles may be distinct from that observed with rigid cargo. Thus, more physiologically relevant studies have used liposomes (25) or isolated vesicles (26) with their in vivo complement of motors (26–28), or even intracellular organelles artificially coupled to a known motor type (17). Here, we attached recombinant myosin Va heavy meromyosin (myoVa) molecules at varying motor density to 100- to 650-nm lipid vesicles with membrane characteristics that were either fluid- or gel-like. When vesicles composed of fluid membranes are transported by an ensemble of myoVa molecules, they demonstrate velocities exceeding both those of identical vesicles transported by a single motor and those of individual, unloaded motors. Gel-like vesicles demonstrate velocities equal to or slower than a single motor. We developed a simulation of vesicular transport that supports at least one simple model wherein reductions in velocity can be attributed to two distinct mechanisms of intermotor interference (i.e., load-dependent modulation of stepping kinetics and binding-site exclusion), whereas enhanced transport velocities result from the concerted effects of myoVa’s stepping and the preferential detachment of the trailing motor in the ensemble from the actin track. This proposed bias in motor detachment and subsequent recentering of the vesicle above the remaining motors would generate an additional forward-directed displacement of the vesicle, providing the additional vesicular velocity. Therefore, we propose that the physical properties of the cargo itself contribute to the emergent transport behavior.     

Bioactive cell-like hybrids coassembled from (glyco)dendrimersomes with bacterial membranes

A library of amphiphilic Janus dendrimers including two that are fluorescent and one glycodendrimer presenting lactose were used to construct giant dendrimersomes and glycodendrimersomes. Coassembly with the components of bacterial membrane vesicles by a dehydration–rehydration process generated giant cell-like hybrid vesicles, whereas the injection of their ethanol solution into PBS produced monodisperse nanometer size assemblies. These hybrid vesicles contain transmembrane proteins including a small membrane protein, MgrB, tagged with a red fluorescent protein, lipopolysaccharides, and glycoproteins from the bacterium Escherichia coli. Incorporation of two colored fluorescent probes in each of the components allowed fluorescence microscopy to visualize and demonstrate coassembly and the incorporation of functional membrane channels. Importantly, the hybrid vesicles bind a human galectin, consistent with the display of sugar moieties from lipopolysaccharides or possibly glycosylated membrane proteins. The present coassembly method is likely to create cell-like hybrids from any biological membrane including human cells and thus may enable practical application in nanomedicine.Naturally occurring (1), chemically modified (2, 3), and synthetic (4, 5) lipids, amphiphilic block copolymers (6, 7), polypeptides (8), Janus dendrimers (JDs) (9), and Janus glycodendrimers (JGDs) (10, 11) self-assemble into vesicles denoted as liposomes, polymersomes, dendrimersomes (DSs), and glycodendrimersomes (GDSs), respectively. These vesicles provide models for primitive (12) and contemporary (13, 14) cell membranes and drug-delivery devices (15–17). Recently, hybrid vesicles coassembled from naturally occurring phospholipids and amphiphilic block copolymers (18–20) have been described; these vesicles eliminated some of the deficiencies of liposomes, such as limited stability under oxidative conditions and general instability over time, and the deficiencies of polymersomes, which possess wide membrane thickness [8–50 nm (20)], exhibit toxicity, and can be tedious to synthesize. These hybrid vesicles combined the desirable feature of liposomes—specifically, their biologically suitable membrane thickness of 4 nm—with that of polymersomes, which are known for their stability. In addition, transmembrane proteins (21–23) could be incorporated into the phospholipid fragments of planar membranes derived from these assemblies. However, the variability in the extent of miscibility between the hydrophobic fragments of the phospholipid and the block copolymer (20) generates a complex morphology of the hybrid membrane that requires further characterization to enable practical applications both as drug-delivery devices and cell membrane models. Here, we report the coassembly of the components of DSs and GDSs with those of the bacterial membrane vesicles (BMVs) to generate functional hybrid vesicles. DSs, GDSs, and liposomes have hydrophobic fragments with similar chemical structures and similar membrane thickness (4.5–4.9 nm) (24). Therefore, the bacterial membranes with their intact native components are expected to be transferred to the hybrid vesicles, providing a new and simple method for the generation of bioactive cell-like hybrids of interest as critical nanoscale design parameters (25).     

Reprogramming of G protein-coupled receptor recycling and signaling by a kinase switch

The postendocytic recycling of signaling receptors is subject to multiple requirements. Why this is so, considering that many other proteins can recycle without apparent requirements, is a fundamental question. Here we show that cells can leverage these requirements to switch the recycling of the beta-2 adrenergic receptor (B2AR), a prototypic signaling receptor, between sequence-dependent and bulk recycling pathways, based on extracellular signals. This switch is determined by protein kinase A-mediated phosphorylation of B2AR on the cytoplasmic tail. The phosphorylation state of B2AR dictates its partitioning into spatially and functionally distinct endosomal microdomains mediating bulk and sequence-dependent recycling, and also regulates the rate of B2AR recycling and resensitization. Our results demonstrate that G protein-coupled receptor recycling is not always restricted to the sequence-dependent pathway, but may be reprogrammed as needed by physiological signals. Such flexible reprogramming might provide a versatile method for rapidly modulating cellular responses to extracellular signaling.How proteins are sorted in the endocytic pathway is a fundamental question in cell biology. This is especially relevant for signaling receptors, given that relatively small changes in rates of receptor sorting into the recycling pathway can cause significant changes in surface receptors, and hence in cellular sensitivity (1–3). Our knowledge of receptor signaling and trafficking comes mainly from studying examples such as the beta-2 adrenergic receptor (B2AR), a prototypical member of G protein-coupled receptor (GPCR) family, the largest family of signaling receptors (2–5). B2AR activation initiates surface receptor removal and transport to endosomes, causing cellular desensitization (6, 7). The rate and extent of resensitization is then determined by B2AR surface recycling (1–3, 8, 9).Interestingly, the recycling of signaling receptors is functionally distinct from the recycling of constitutively cycling proteins like the transferrin receptor (TfR) (1, 6, 10, 11). TfR recycles by “bulk” geometric sorting, largely independent of specific cytoplasmic sequences (12, 13). B2AR recycling, in contrast, requires a specific PSD95-Dlg1-zo-1 domain (PDZ)-ligand sequence on its C-terminal tail, which links the receptor to the actin cytoskeleton (14, 15). Recent work has identified physically and biochemically distinct microdomains on early endosomes that mediate B2AR recycling independent of TfR (14–16). Although the exact mechanisms of B2AR sorting into these domains remain under investigation, this sorting clearly requires specific sequence elements on B2AR (1, 10, 11, 17). Importantly, why signaling receptor sorting is subject to such specialized requirements, considering that cargo like TfR apparently can recycle without specific sequence requirements, is not clear (1, 12–16). One possibility is that these requirements allow signaling pathways to regulate and redirect receptor trafficking between different pathways as needed (17–19). Although this is an attractive idea, whether and how physiological signals regulate receptor sorting remain poorly understood (7, 19).Here we show that adrenergic signaling can switch B2AR recycling between the sequence-dependent and bulk recycling pathways. Adrenergic activation, via protein kinase A (PKA)-mediated B2AR phosphorylation on the cytoplasmic tail, restricts B2AR to spatially defined PDZ- and actin-dependent endosomal microdomains. Dephosphorylation of B2AR switches B2AR to the bulk (PDZ-independent) recycling pathway, causing faster recycling of B2AR and increased cellular sensitivity. Our results suggest that cells may leverage sequence requirements for rapid adaptive reprogramming of signaling receptor trafficking and cellular sensitivity.     

CK2 activates kinesin via induction of a conformational change

Kinesin is the canonical plus-end microtubule motor and has been the focus of intense study since its discovery in 1985. We previously demonstrated a time-dependent inactivation of kinesin in vitro that was fully reversible by the addition of purified casein kinase 2 (CK2) and showed that this inactivation/reactivation pathway was relevant in cells. Here we show that kinesin inactivation results from a conformational change that causes the neck linker to be positioned closer to the motor domain. Furthermore, we show that treatment of kinesin with CK2 prevents and reverses this repositioning. Finally, we demonstrate that CK2 treatment facilitates ADP dissociation from the motor, resulting in a nucleotide-free state that promotes microtubule binding. Thus, we propose that kinesin inactivation results from neck-linker repositioning and that CK2-mediated reactivation results from CK2’s dual ability to reverse this repositioning and to promote ADP release.Intracellular microtubule-based transport is crucial for the creation and maintenance of cellular order, and altered transport is implicated in both neurodegeneration and cancer. Frequently, in vivo cargos are moved by multiple microtubule-based molecular motors (1–6), and changing the number of active motors on the cargo can change cargo force production (4) and also potentially the mean travel distance for predominantly unidirectionally moving cargos (7). However, until recently, it has been unclear how activity of cargo-bound motors might be regulated.Transport is frequently regulated by signaling cascades [see, e.g., cAMP control of pigment granule transport (8) or APP transport (9)]. Thus, multiple signaling pathways might contribute to control of transport under different conditions, and signaling altered in disease might affect transport, which could then contribute to disease progression. Nonetheless, mechanistic understanding of such effects is limited. For these reasons, we would like to understand transport roles of specific disease-relevant kinases. One such kinase is casein kinase 2 (CK2), which is involved in development (10), is up-regulated in various cancers (11), and is decreased in neurodegeneration (12). We found that, over time, kinesin loses its ability to bind microtubules (becomes “inactive”) and that this loss of activity could be reversed by CK2 (13).Mechanistically, how kinesin became inactive—and what CK2 did to reactivate it—was unknown. Here we discover that kinesin’s inactivation results from a conformational change involving repositioning of the neck linker (NL) and that reactivation reverses this conformational change. Intriguingly, the conformational change that results in reactivation causes release of ADP, converting kinesin from a weak microtubule-interacting state (K⋅ADP) to a strong one (K), so that in some ways CK2 acts like a small G-protein nucleotide-exchange factor.