An in vitro vesicle formation assay reveals cargo clients and factors that mediate vesicular trafficking

The fidelity of protein transport in the secretory pathway relies on the accurate sorting of proteins to their correct destinations. To deepen our understanding of the underlying molecular mechanisms, it is important to develop a robust approach to systematically reveal cargo proteins that depend on specific sorting machinery to be enriched into transport vesicles. Here, we used an in vitro assay that reconstitutes packaging of human cargo proteins into vesicles to quantify cargo capture. Quantitative mass spectrometry (MS) analyses of the isolated vesicles revealed cytosolic proteins that are associated with vesicle membranes in a GTP-dependent manner. We found that two of them, FAM84B (also known as LRAT domain containing 2 or LRATD2) and PRRC1, contain proline-rich domains and regulate anterograde trafficking. Further analyses revealed that PRRC1 is recruited to endoplasmic reticulum (ER) exit sites, interacts with the inner COPII coat, and its absence increases membrane association of COPII. In addition, we uncovered cargo proteins that depend on GTP hydrolysis to be captured into vesicles. Comparing control cells with cells depleted of the cargo receptors, SURF4 or ERGIC53, we revealed specific clients of each of these two export adaptors. Our results indicate that the vesicle formation assay in combination with quantitative MS analysis is a robust and powerful tool to uncover novel factors that mediate vesicular trafficking and to uncover cargo clients of specific cellular factors.

The eukaryotic secretory pathway plays important roles in delivering a variety of newly synthesized proteins to their specific resident compartments. The fidelity of protein transport in the secretory pathway depends on accurate sorting of specific cargo proteins into transport vesicles. Defects in cargo sorting cause protein mistargeting and induce defects in establishing cell polarity, immunity, as well as other physiological processes (1).A variety of cytosolic proteins are recruited to the membrane and play important roles in the protein sorting process. These cytosolic proteins include small GTPases of the Arf family and cargo adaptors (1, 2). The Arf family GTPases cycle between a GDP-bound cytosolic state and a GTP-bound state. Upon GTP binding, Arf proteins undergo conformational changes in which the N-terminal amphipathic helix is exposed to bind membranes and the switch domains change their conformation to recruit various cytosolic cargo adaptors. Once recruited onto the membranes, these cargo adaptors recognize sorting motifs on the cargo proteins. This recognition step is important for efficiently capturing cargo proteins into vesicles.The Arf family protein, Sar1, regulates packaging of cargo proteins into vesicles at the endoplasmic reticulum (ER). GTP-bound Sar1 mediates membrane recruitment of the coat protein complex II (COPII) to capture cargo proteins (2). Soluble cargo proteins in the lumen of the ER cannot be directly recognized by COPII coat and such proteins are thought to be linked to the cargo sorting machinery on the cytosolic side by transmembrane cargo receptors. One cargo receptor in mammalian cells, ERGIC53, is a mannose lectin and functions in capturing specific N-linked glycoproteins in the lumen of the ER (3). ERGIC53 regulates ER export of blood coagulation factors V and VIII, a cathepsin-Z–related protein, and alpha1-antittrypsin (4–7). Another cargo receptor, SURF4, binds amino-terminal tripeptide motifs of soluble cargo proteins and regulates ER export of soluble cargo proteins, including the yolk protein VIT-2 in Caenorhabditis elegans (8), and PCSK9 and apolipoprotein B in mammalian cells (9–11).Although significant progress has been made in understanding the general steps of cargo sorting, the spectrum of cargo clients of a specific Arf family member, cargo adaptor, or cargo receptor remains largely underinvestigated. To deepen our understanding of protein sorting in the secretory pathway, it is important to develop a robust approach to systematically reveal cargo proteins that depend on a specific factor to be efficiently packaged into vesicles. Revealing this will provide significant insight into the functions and the specificity of cargo sorting. Since distinct cytosolic proteins are recruited to membranes by different GTP-bound Arf family proteins, systematic approaches are needed to characterize budding events associated with a specific GTP-bound Arf family protein.A cellular imaging approach, pairing analysis of cargo receptors (PAIRS), has been utilized to identify the spectrum of cargo proteins that depend on a specific cargo receptor for ER export in yeast. This analysis focused on around 150 cargo molecules labeled with fluorescent tags (12). An in vitro assay that reconstitutes packaging of cargo proteins into vesicles has been used to reveal protein profiles of vesicles budded with purified COPII or COPI proteins (13). However, this analysis did not identify any non-ER resident transmembrane proteins or secretory proteins (13). This is possibly due to an unappreciated requirement for other cytosolic factors in addition to the COP coats. Affinity chromatography has been utilized to reveal cytosolic proteins that specifically interact with GTP-bound Arf or Rab proteins (14–16). In this approach, the membranes are disrupted, which might preclude identification of membrane-associated effectors. Thus, it is important to develop additional approaches to reveal novel cytosolic proteins that associate with GTP-bound Arf proteins on membranes.Here, we used an in vitro assay to reconstitute packaging of cargo proteins into transport vesicles utilizing rat liver cytosol (RLC) as a source of cytosolic proteins. Analysis of vesicle fractions by quantitative mass spectrometry (MS) revealed cytosolic proteins that are associated with vesicles dependent on GTP or GTP-bound Sar1A, and that regulate protein trafficking. One of the identified proteins, PRRC1, regulates membrane association of the COPII coat and facilitates ER-to-Golgi trafficking. We also revealed cargo proteins that depend on specific cargo receptors, ERGIC53 or SURF4, to be efficiently packaged into vesicles. Our study indicates that the vesicle formation assay is a robust tool to reveal functional roles of specific factors in protein sorting, and to uncover novel factors that regulate vesicular trafficking in the secretory pathway.     

Selective inhibition of protein secretion by abrogating receptor–coat interactions during ER export

Protein secretion is an essential process that drives cell growth, movement, and communication. Protein traffic within the secretory pathway occurs via transport intermediates that bud from one compartment and fuse with a downstream compartment to deliver their contents. Here, we explore the possibility that protein secretion can be selectively inhibited by perturbing protein–protein interactions that drive capture into transport vesicles. Human proprotein convertase subtilisin/kexin type 9 (PCSK9) is a determinant of cholesterol metabolism whose secretion is mediated by a specific cargo adaptor protein, SEC24A. We map a series of protein–protein interactions between PCSK9, its endoplasmic reticulum (ER) export receptor SURF4, and SEC24A that mediate secretion of PCSK9. We show that the interaction between SURF4 and SEC24A can be inhibited by 4-phenylbutyrate (4-PBA), a small molecule that occludes a cargo-binding domain of SEC24. This inhibition reduces secretion of PCSK9 and additional SURF4 clients that we identify by mass spectrometry, leaving other secreted cargoes unaffected. We propose that selective small-molecule inhibition of cargo recognition by SEC24 is a potential therapeutic intervention for atherosclerosis and other diseases that are modulated by secreted proteins.

Approximately a third of human protein-coding genes encode proteins that enter the secretory pathway. Such secretory proteins include integral membrane proteins, soluble proteins located in the lumen of membrane-bound organelles, and proteins that are transported outside of the cell. The vast majority of secretory proteins enter the pathway at the endoplasmic reticulum (ER), where they reach their mature state after acquiring posttranslational modifications and interacting with the abundant chaperones of the ER lumen. Secretion-competent mature proteins are exported from the ER via vesicles generated by the COPII (coat protein complex II) coat and delivered to the Golgi apparatus, where they are further modified (1). Vesicle biogenesis occurs via self-assembly of the COPII coat at ER exit sites (ERESs). Protein capture into COPII vesicles occurs via two mechanisms: signal-mediated export and bulk flow (2). In signal-mediated export, specific signals within cargo proteins are recognized by the COPII coat, which then concentrates cargo at sites of vesicle formation. The coat–cargo interaction is direct for many transmembrane proteins, whereas soluble proteins or proteins that do not expose cytoplasmic signals require export receptors that bridge the interaction between cargo and coat (3). In contrast, bulk flow export occurs in the absence of signals and relies on diffusion of a cargo molecule into an ERES followed by passive uptake into a nascent vesicle (4, 5). The extent to which signal-mediated versus bulk flow mechanisms are used for the entire secretome remains to be fully determined.The COPII coat subunit, Sec24, is the cargo adaptor that confers selective cargo capture and enrichment during vesicle formation. Mutagenesis and structural studies have revealed multiple cargo-binding sites on Sec24 (6–8), which permits recognition of diverse cargoes by a single coat subunit (3). The repertoire of cargo molecules recognized by the coat is further expanded by multiple isoforms of Sec24: Saccharomyces cerevisiae express three Sec24 homologs (Sec24, Iss1/Sfb2, and Lst1/Sfb3); humans express four isoforms (SEC24A–D). SEC24A and SEC24B share 60% sequence identity, and SEC24C and SEC24D similarly share more than 50% sequence identity. Accordingly, the four isoforms can be classified into two subfamilies, SEC24A/B and SEC24C/D, that share nonoverlapping recognition specificity (9–12).The specificity of Sec24-mediated cargo recognition raises the possibility that secretion, an essential process, might be selectively targeted by preventing a subset of Sec24–cargo interactions. Indeed, release of the secreted proprotein convertase subtilisin/kexin type 9 (PCSK9) was selectively perturbed in SEC24A knockout(KO) mice (13). Because PCSK9 participates in cholesterol homeostasis, the predominant physiological effect of SEC24A KO was reduced circulating cholesterol, suggesting a novel therapeutic avenue for cardiovascular health. Here, we sought to probe the network of protein–protein interactions that contribute to PCSK9 secretion with the goal of understanding which interactions might be perturbed to achieve a therapeutic outcome.PCSK9 secretion via SEC24A is facilitated by the ER export receptor SURF4, which also mediates secretion of multiple calcium-binding extracellular matrix proteins that employ N-terminal ϕ-P-ϕ signals, where ϕ is any hydrophobic amino acid (14, 15). This motif has been termed the ER-ESCAPE (ER-Exit by Soluble Cargo using Amino-terminal Peptide-Encoding) motif (15). The precise mechanism by which SURF4 recognizes this signal remains unknown, and the nature of the interaction between SURF4 and SEC24A also remains unclear. Here, we demonstrate that the SEC24A–SURF4 interaction is driven by a conserved cargo-binding domain on SEC24A known as the B-site (7, 8), which can be occluded by small-molecule treatment (16) to selectively inhibit secretion of PCSK9. Moreover, we identify additional clients of SURF4 that, like PCSK9, use N-terminal ER-ESCAPE motifs to achieve SURF4-dependent secretion. The network of client–receptor–adaptor interactions revealed by our study lends support to the concept of selective inhibition of protein secretion as a therapeutic target.     

Gas1 is a receptor for sonic hedgehog to repel enteric axons

The myenteric plexus of the enteric nervous system controls the movement of smooth muscles in the gastrointestinal system. They extend their axons between two peripheral smooth muscle layers to form a tubular meshwork arborizing the gut wall. How a tubular axonal meshwork becomes established without invading centrally toward the gut epithelium has not been addressed. We provide evidence here that sonic hedgehog (Shh) secreted from the gut epithelium prevents central projections of enteric axons, thereby forcing their peripheral tubular distribution. Exclusion of enteric central projections by Shh requires its binding partner growth arrest specific gene 1 (Gas1) and its signaling component smoothened (Smo) in enteric neurons. Using enteric neurons differentiated from neurospheres in vitro, we show that enteric axon growth is not inhibited by Shh. Rather, when Shh is presented as a point source, enteric axons turn away from it in a Gas1-dependent manner. Of the Gαi proteins that can couple with Smo, G protein α Z (Gnaz) is found in enteric axons. Knockdown and dominant negative inhibition of Gnaz dampen the axon-repulsive response to Shh, and Gnaz mutant intestines contain centrally projected enteric axons. Together, our data uncover a previously unsuspected mechanism underlying development of centrifugal tubular organization and identify a previously unidentified effector of Shh in axon guidance.The enteric nervous system is the largest peripheral nervous subsystem, often likened to a second brain (1). Embedded in the gastrointestinal tract, it controls various aspects of digestive function ranging from movement and secretion to absorption. Despite their final location, enteric neurons are of neural crest origin, and the majority of enteric progenitors are derived from vagal neural crest cells at midgestation stages of the mouse embryo (1, 2). Under the control of glial cell-derived neurotrophic factor (GDNF) and Endothelin 3 (END3) signaling, they migrate into the anterior gut mesenchyme. Once there, they continue to propagate and migrate posteriorly through most of the length of the gut. The first appearing enteric arbor is the myenteric plexus, composed of islands of neurons interconnected by longitudinal and circumferential axons between the inner circular and outer longitudinal smooth muscle layers in the stomach, intestine, and colon to effect contraction. In cross-section, this plexus presents a simple centrifugal ring structure, but when viewed superficially, it is organized as an extensive mesh-like tubular arborization encasing the gut wall. Axonal arborization along the gut smooth muscles is controlled by overlapping and sequential actions of GDNF and Neuturin, a GDNF family member acting mainly postnatally (3, 4). Recently, the planar polarity pathway has also been implicated in enteric connectivity (5). Patients carrying mutations that cause enteric neuron deficiency, including in genes of the GDNF signaling pathway, present with megacolon and constipation associated with poor bowel movement, a condition known as Hirschsbrung’s disease (1, 2).Growth of the gut mucosal mesenchyme and smooth muscle, on the other hand, depends on Hedgehog (Hh) signaling during embryonic development (6–8). Both Sonic Hh (Shh) and Indian Hh (Ihh) are expressed in the gut endoderm (6), which is radially surrounded by mucosa mesenchyme, smooth muscles, and enteric neurons (1, 2). Germ-line and endoderm-specific conditional mouse mutants for Shh and/or Ihh display reduced mesenchyme and smooth muscle mass (6, 8). Shh and Ihh directly act on the gut mesoderm, as inactivation of smoothened (Smo), the obligatory Hh signaling component, in gut mesoderm causes the same defect (8). Mice mutant for growth arrest specific gene 1 (Gas1), which encodes an Hh surface binding receptor (9, 10), also share this defect (11).The role of Hh signaling in enteric neuron development is less defined. Shh mutant intestines contained more punctuated Tuj1 (an antibody recognizing neuronal βΙΙΙ-tubulin) staining signals erroneously located near the base of the villi and were interpreted to have a substantial number of mislocalized enteric neurons, whereas Ihh mutants had no enteric neurons (6). However, endoderm-specific conditional Shh;Ihh mutants were reported to have enteric neurons (8). Using the enteric progenitor/neuron marker P75 to better assess the cell body, we showed that Gas1 mutant intestines had quantitatively more P75-positive (P75⁺) enteric progenitors/neurons due to increased proliferation, and a small fraction of them were abnormally located near the base of the villi (11). However, the enteric abnormalities of Shh and Gas1 mutants may arise as a secondary patterning defect (12) due to reduced mesenchyme/smooth muscle mass or reduced levels of BMP4 in the mesenchyme (6), as ectopic expression of BMP4 could negatively influence enteric progenitor positioning and proliferation (12). Conversely, recombinant Shh can inhibit enteric progenitor differentiation and migration in vitro (7). Single nucleotide polymorphisms (SNPs) of the PTCH1 gene, a conserved Hh pathway inhibitory component, are associated with a high risk of Hirschsbrung’s disease (13, 14). Furthermore, Ptch1 conditional mutant mouse enteric progenitors have a reduced propensity for neurogenesis in vitro (13). Thus, Shh signaling may play a direct role in enteric neuron development.Whether the gut epithelium directly acts on the myenteric axons to confine their periphery connectivity within the smooth muscles is not known. During our continuous investigation of Shh and Gas1 mutant gut defects, we found that their intestines contained Tuj1 staining signals deep inside the villi, almost reaching the gut epithelium, suggesting a previously unappreciated axonal projection defect. Given that Shh is an axon guidance molecule in the central nervous system (CNS) (15, 16), we were motivated to investigate whether Shh in the gut epithelium acts to prevent erroneous entry of enteric axons into the villi. Below, we provide evidence for a previously unknown enteric axon repulsion mechanism mediated directly by Shh via its receptor Gas1 and signaling component Smo. We further implicate a unique Gαi protein, the pertussis toxin (PTX)-insensitive Gnaz (17), in mediating Shh-directed chemorepulsion.     

ER-phagy requires the assembly of actin at sites of contact between the cortical ER and endocytic pits

Fragments of the endoplasmic reticulum (ER) are selectively delivered to the lysosome (mammals) or vacuole (yeast) in response to starvation or the accumulation of misfolded proteins through an autophagic process known as ER-phagy. A screen of the Saccharomyces cerevisiae deletion library identified end3Δ as a candidate knockout strain that is defective in ER-phagy during starvation conditions, but not bulk autophagy. We find that loss of End3 and its stable binding partner Pan1, or inhibition of the Arp2/3 complex that is coupled by the End3-Pan1 complex to endocytic pits, blocks the association of the cortical ER autophagy receptor, Atg40, with the autophagosomal assembly scaffold protein Atg11. The membrane contact site module linking the rim of cortical ER sheets and endocytic pits, consisting of Scs2 or Scs22, Osh2 or Osh3, and Myo3 or Myo5, is also needed for ER-phagy. Both Atg40 and Scs2 are concentrated at the edges of ER sheets and can be cross-linked to each other. Our results are consistent with a model in which actin assembly at sites of contact between the cortical ER and endocytic pits contributes to ER sequestration into autophagosomes.

More than a third of the proteome translocates across the endoplasmic reticulum (ER) membrane and enters the ER lumen in an unfolded state (1). Even under normal conditions, a significant fraction of newly translocated polypeptides fails to properly fold (2). This can interfere with ER functions, including the folding and export of other proteins (3, 4). The degradation of compromised sections of the ER can occur by several different mechanisms. Here we focus on a type of selective autophagy known as macro-ER-phagy (hereafter ER-phagy) (5) that is induced by starvation or rapamycin treatment (5, 6). The ability to recycle components of the ER and to reduce the biosynthetic capacity of the ER to match a decrease in cellular demands is important for homeostasis. The mechanisms by which regions of the ER are recognized, severed from the ER network, and processed for degradation through ER-phagy are incompletely understood.The ER is comprised of several distinct yet contiguous regions that together form one structure extending throughout the cell volume (7, 8). In many cell types, a polygonal network of smooth ER tubules is found at the cell periphery, while ribosome-studded ER sheets occupy a more central region of the cell. The outer nuclear membrane is also contiguous with the ER and shares many of its functions. Different degradation pathways have been implicated in the turnover of these different regions (9). ER-phagy involves the sequestration of ER segments by autophagosomes, which after closure deliver their contents into the lysosome (the vacuole in yeast) for degradation. This process utilizes ER-associated cargo receptors that allow selective recognition and incorporation into autophagosomes. Mammalian cells employ different ER-phagy receptors for the selective degradation of ER sheets and tubules (10). In the yeast Saccharomyces cerevisiae, Atg40 is the cargo receptor for ER-phagy of the cortical ER (cER), ER that is juxtaposed to the plasma membrane (PM), while Atg39 is the cargo receptor for ER-phagy of the nuclear envelope (11). All autophagy receptors bind to Atg8 (yeast), LC3, or GABARAP proteins (mammals) (10, 12). Here we have focused on Atg40-dependent cER-phagy. This pathway can be induced by the TOR inhibitor rapamycin, starvation, or the accumulation of aggregation-prone proteins (11, 13). Atg40 shares similarities with two different mammalian ER-phagy receptors, the FAM134 protein family and RTN3L (11, 14, 15). Atg40 has the same domain structure as the ER sheets receptor FAM134B (11, 14), yet it primarily localizes to ER tubules like RTN3L (11, 15, 16). Atg40, FAM134 proteins, and RTN3L all contain reticulon homology domains (11, 14, 15).Other than ER-phagy receptors, only a few additional components have been implicated in recognizing, severing and sequestering fragments of the ER for degradation. Recent studies have demonstrated a noncanonical role for the COPII vesicle coat subunit, Lst1 (SEC24C in mammals), in ER-phagy. Lst1, which forms a complex with Sec23, works in conjunction with Atg40 to select ER subdomains for degradation (13). Additionally, Lnp1, a protein that promotes remodeling of the cER network by stabilizing newly formed three-way junctions (17, 18), is required for cER-phagy (19). The roles of Lst1 and Lnp1 in ER-phagy appear to be evolutionarily conserved (20). Inhibition of actin polymerization with the drug latrunculin A (LatA) blocks ER-phagy (6) as well as other selective autophagy pathways in yeast (21). LatA also prevents the association of Atg40 with the autophagosome assembly scaffold protein, Atg11 (19). This step precedes the sequestration of the ER by autophagosomes at the phagophore assembly site (PAS), perivacuolar sites where autophagosomes form (22). These observations suggest that actin-dependent ER remodeling and/or transport may be needed to bring elements of the cER carrying Atg40 to the PAS.In an effort to identify additional components of the ER-phagy machinery, we performed a systematic screen for mutants defective in the selective autophagic turnover of the cER. This screen led to the identification of a number of candidates, including the VPS13 gene (23). VPS13 encodes a protein that mediates phospholipid transfer at interorganelle contact sites (23, 24). In the absence of Vps13, the late endosome enlarges. Interestingly, the loss of Vps13 does not disrupt the association of Atg40 with Atg11, but instead blocks the subsequent sequestration of ER into autophagosomes (23). Understanding the components and mechanisms involved in ER-phagy could also reveal new approaches to treat human disease conditions in which ER function is impaired. A number of the components involved in ER-phagy have been implicated in several pathologies, including different forms of hereditary spastic paraplegias and Parkinson’s disease (25). Here, we have pursued another candidate from our initial screen, and this has led us to investigate the role of actin assembly components in cER-phagy.     

Stromal response to Hedgehog signaling restrains pancreatic cancer progression

Pancreatic ductal adenocarcinoma (PDA) is the most lethal of common human malignancies, with no truly effective therapies for advanced disease. Preclinical studies have suggested a therapeutic benefit of targeting the Hedgehog (Hh) signaling pathway, which is activated throughout the course of PDA progression by expression of Hh ligands in the neoplastic epithelium and paracrine response in the stromal fibroblasts. Clinical trials to test this possibility, however, have yielded disappointing results. To further investigate the role of Hh signaling in the formation of PDA and its precursor lesion, pancreatic intraepithelial neoplasia (PanIN), we examined the effects of genetic or pharmacologic inhibition of Hh pathway activity in three distinct genetically engineered mouse models and found that Hh pathway inhibition accelerates rather than delays progression of oncogenic Kras-driven disease. Notably, pharmacologic inhibition of Hh pathway activity affected the balance between epithelial and stromal elements, suppressing stromal desmoplasia but also causing accelerated growth of the PanIN epithelium. In striking contrast, pathway activation using a small molecule agonist caused stromal hyperplasia and reduced epithelial proliferation. These results indicate that stromal response to Hh signaling is protective against PDA and that pharmacologic activation of pathway response can slow tumorigenesis. Our results provide evidence for a restraining role of stroma in PDA progression, suggesting an explanation for the failure of Hh inhibitors in clinical trials and pointing to the possibility of a novel type of therapeutic intervention.Pancreatic ductal adenocarcinoma (PDA) is the fourth most common cause of cancer-related death in the United States and is the most lethal of common human malignancies, with a 5-y survival rate of ∼7% (1, 2). The most effective chemotherapy regimens for metastatic or locally advanced inoperable disease are largely palliative and are capable of extending overall survival by only several months (3, 4). Even localized disease, treatable with surgery followed by adjuvant chemotherapy, has a dismal 5-y survival rate of 24% (1). Among gastrointestinal malignancies, PDA is unique in that it is predominantly driven by oncogenic Kras activity. In addition, PDA pathogenesis is marked by a striking desmoplastic reaction to invading tumor cells. This desmoplasia includes a dense extracellular matrix with abundant stromal fibroblasts and influences the cellular biology of the tumor as well as its response to chemotherapeutic agents.Hedgehog (Hh) signaling has been thought to play a role in PDA desmoplasia and tumor progression but is notable during embryonic development of the pancreas for its absence in the region of embryonic endoderm from which the pancreas forms (5–7). This absence of activity is required for normal specification of early pancreatic progenitor fate, and pharmacologic or antibody treatments that inhibit Hh pathway signaling cause expansion of such fates, as indicated by ectopic expression of the pancreatic progenitor marker Pdx1 (5–7). In adult mice, expression of Shh (Sonic hedgehog) is barely detectable and is limited to pancreatic duct glands, which are small outpouchings of the major pancreatic ducts (8).Increased levels of Shh expression are observed in the setting of pancreatic injury and throughout the course of human and murine PDA progression, beginning in the early epithelial precursor lesions, acinar-ductal metaplasia (ADM) and pancreatic intraepithelial neoplasia (PanIN). Shh is also expressed in locally invasive carcinomas and in distant metastases (9–11). Pharmacologic blockade of Hh pathway response with antagonists, such as cyclopamine and HhAntag, that inhibit activity of the critical Hh-transducing molecule Smoothened (Smo) has been reported to reduce the growth of human pancreatic cancer xenografts in nude mice (9, 11, 12); cyclopamine was also reported to prolong survival in a genetically engineered mouse (GEM) model of pancreatic cancer (13). Hh pathway blockade using either small-molecule antagonists or the Shh ligand-blocking antibody 5E1 was also reported to inhibit distant metastases from human pancreatic xenografts in athymic nude mice (14–16).Hh signaling in normal pancreas and in PDA is exclusively paracrine (17), with expression of Shh limited to epithelium and response restricted to stroma. Correspondingly, deletion of Smo in the pancreatic epithelium does not affect PDA pathogenesis in a GEM model (18). Hh response and its inhibition thus primarily affect stromal cells and, in the setting of PDA, has been reported to have a major impact on the desmoplastic reaction (19–21). An indirect therapeutic benefit of Hh pathway blockade thus may be to decrease stromal fibrosis and increase functional vascularity, potentially enhancing the penetration and effectiveness of standard chemotherapy (20).Given the preclinical evidence suggesting possible therapeutic benefits of Hh pathway blockade in limiting local or metastatic PDA growth and enhancement of chemotherapy, several clinical trials have been launched using small-molecule Hh pathway antagonists for this disease (22). These trials have typically combined an Hh pathway antagonist with standard chemotherapy, but, unfortunately, results have been either negative or equivocal. Thus, for example, in a phase 2 double-blind placebo-controlled study of saridegib, a cyclopamine derivative, 122 patients with previously untreated metastatic PDA were treated with either saridegib plus gemcitabine or placebo plus gemcitabine, with overall survival (OS) as a primary end point. Interim data analysis indicated that median OS for the saridegib plus gemcitabine arm was less than 6 mo whereas the median OS for the placebo plus gemcitabine arm was greater than 6 mo, resulting in termination of the clinical trial (23). In another randomized, placebo-controlled phase 2 study, the FDA-approved Smo antagonist vismodegib plus gemcitabine was compared with placebo plus gemcitabine in patients with previously untreated metastatic PDA (24). At the time of interim analysis, the OS was 6.3 versus 5.4 mo for vismodegib versus the placebo arm, with an unimpressive hazard ratio of 0.97. Recently, an interim analysis was reported of a single-arm phase 2 study using vismodegib in combination with gemcitabine and nab-paclitaxel (25), with an estimated OS of 10 mo for 59 patients, which is greater than the published historic controls of 8.5 mo for gemcitabine plus nab-paclitaxel (4).     

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.     

In situ architecture of the lipid transport protein VPS13C at ER–lysosome membrane contacts

VPS13 is a eukaryotic lipid transport protein localized at membrane contact sites. Previous studies suggested that it may transfer lipids between adjacent bilayers by a bridge-like mechanism. Direct evidence for this hypothesis from a full-length structure and from electron microscopy (EM) studies in situ is still missing, however. Here, we have capitalized on AlphaFold predictions to complement the structural information already available about VPS13 and to generate a full-length model of human VPS13C, the Parkinson’s disease–linked VPS13 paralog localized at contacts between the endoplasmic reticulum (ER) and endo/lysosomes. Such a model predicts an ∼30-nm rod with a hydrophobic groove that extends throughout its length. We further investigated whether such a structure can be observed in situ at ER–endo/lysosome contacts. To this aim, we combined genetic approaches with cryo-focused ion beam (cryo-FIB) milling and cryo–electron tomography (cryo-ET) to examine HeLa cells overexpressing this protein (either full length or with an internal truncation) along with VAP, its anchoring binding partner at the ER. Using these methods, we identified rod-like densities that span the space separating the two adjacent membranes and that match the predicted structures of either full-length VPS13C or its shorter truncated mutant, thus providing in situ evidence for a bridge model of VPS13 in lipid transport.

Communication between subcellular membranous organelles mediated by direct contacts not leading to their fusion plays a critical role in the physiology of eukaryotic cells and is an important complement to interorganelle communication mediated by vesicular transport. The endoplasmic reticulum (ER), in particular, establishes direct contacts with all other membranous organelles of the cell (1). One of the functions of such contacts is to mediate lipid transfer between the two closely apposed membranes via proteins that contain lipid transport modules and also act as direct or indirect tethers between them (2–4). Until few years ago, lipid transfer proteins acting at membrane contact sites in eukaryotic cells were thought to act exclusively by a shuttle mechanism, that is, via modules that harbor one or few lipids and shuttle back and forth between the two participating membranes (5). However, recent studies have also suggested the occurrence of proteins that provide hydrophobic bridges along which lipids can move directly from one bilayer to another to achieve bulk lipid transport (5, 6).The founding member of this class of protein is VPS13, a protein first identified in yeast by genetic screens for membrane traffic proteins (7–9). Subsequently, VPS13 was putatively linked to lipid transport because of the identification of VPS13 dominant mutations as suppressors of the deficiency of ERMES (ER–mitochondria encounter structure), a protein complex that mediates lipid transport between the ER and mitochondria in yeast (10, 11). The human genome comprises four VPS13 proteins, referred to as VPS13A, VPS13B, VPS13C, and VPS13D, which have different localizations at membrane contact sites and whose mutations result in neurodevelopmental defects or neurodegenerative conditions (12–15). A role of VPS13 family proteins in lipid transport, and more specifically at membrane contact sites, has now been supported by genetic, biochemical, imaging, and structural studies (6, 10, 11, 16–21). Fragments of VPS13 were shown to contain multiple lipids and to transfer lipids between artificial liposomes (17). Crystallographic studies of the N-terminal region of VPS13 of Chaetomium thermophilum revealed a hydrophobic cavity (17), which cryo-EM studies of a longer rod-like fragment of the same protein subsequently suggested to continue as a hydrophobic groove along the entire length of the fragment (19). Moreover, binding sites for the ER and for other organelles have been identified in the N-terminal and C-terminal regions of several VPS13 family members, respectively (17, 18, 20–22). Together, these results have suggested a bridge model of lipid transport ideally suited for the bulk delivery of lipids and in line with some of the proposed functions of VPS13 family members in membrane expansion (6). The identification of regions of amino acid similarity and of predicted structural similarities between VPS13 and the autophagy factor ATG2 had suggested a similar function of ATG2 (17, 23, 24).Despite these studies, direct evidence for an arrangement of VPS13–ATG2 family proteins in situ that would support a bridge model of lipid transport is still missing. The goal of this study was to fill this gap by combining cryo-focused ion beam (cryo-FIB) milling and cryo-electron tomography (cryo-ET) imaging, a recently developed method that allows us to resolve the in situ structure of proteins of interest at subnanometer resolution (25–27). To this end, we focused on VPS13C, the VPS13 isoform that is localized at ER–late endosome/lysosome (endo/lysosome) contact sites. VPS13C binds the ER via an FFAT motif-dependent interaction with the ER protein VAP and endo/lysosomal membranes via an interaction with Rab7 (17). Overexpression of VPS13C and VAP results in the extensive formation or ER–endo/lysosome contacts, thus facilitating their identification. Our cryo-ET analysis of cell cryo-lamellae generated by cryo-FIB milling provides in situ structural evidence to support the bridge model of lipid transport and also suggests a dynamic interaction of VPS13C with the ER bilayer. VPS13C loss of function results in lysosomal dysfunction at the cellular level (21) and causes familial Parkinson’s disease in human patients (14). Moreover, as dysfunction of other VPS13 family also results in neurological diseases (28), a better understanding of in situ architecture VPS13C has implications for both biology and medicine.     

From the Cover: Feature Article: mTOR complex 2-Akt signaling at mitochondria-associated endoplasmic reticulum membranes (MAM) regulates mitochondrial physiology

The target of rapamycin (TOR) is a highly conserved protein kinase and a central controller of growth. Mammalian TOR complex 2 (mTORC2) regulates AGC kinase family members and is implicated in various disorders, including cancer and diabetes. Here we report that mTORC2 is localized to the endoplasmic reticulum (ER) subcompartment termed mitochondria-associated ER membrane (MAM). mTORC2 localization to MAM was growth factor-stimulated, and mTORC2 at MAM interacted with the IP₃ receptor (IP3R)-Grp75–voltage-dependent anion-selective channel 1 ER-mitochondrial tethering complex. mTORC2 deficiency disrupted MAM, causing mitochondrial defects including increases in mitochondrial membrane potential, ATP production, and calcium uptake. mTORC2 controlled MAM integrity and mitochondrial function via Akt mediated phosphorylation of the MAM associated proteins IP3R, Hexokinase 2, and phosphofurin acidic cluster sorting protein 2. Thus, mTORC2 is at the core of a MAM signaling hub that controls growth and metabolism.Mitochondria-associated endoplasmic reticulum (ER) membrane (MAM) is a subcompartment of the ER that forms a quasisynaptic structure with mitochondria. The main function of this membrane is to facilitate the transfer of lipids and calcium between the two organelles. MAM thereby controls mitochondrial physiology and apoptosis (1, 2). MAM also mediates ER homeostasis and lipid biosynthesis by harboring chaperones and several key lipid synthesis enzymes (3–6). In mammalian MAM, the ER and mitochondria are physically tethered to each other by the IP₃ receptor (IP3R)-Grp75-VDAC1 (voltage-dependent anion-selective channel 1) trimeric complex (7) and by dimers of the mitofusin (Mfn) proteins Mfn1 and Mfn2 (8) (Fig. S1H). The σ-1 receptor also stabilizes MAM by interacting with IP3R and VDAC (9). MAM formation is regulated by multiple signaling inputs, including calcium and possibly growth factors (10–12). However, the mechanism(s) that controls MAM formation is largely unknown other than it involves recruitment of MAM components by the MAM resident proteins phosphofurin acidic cluster sorting protein 2 (PACS2) and Rab32 (13–15). Akt, an AGC family kinase that is also found at MAM (16), phosphorylates PACS2 (17), but it remains to be determined whether Akt is involved in mediating MAM integrity.Akt, often up-regulated in cancer, also phosphorylates hexokinase 2 (HK2) to promote association of HK2 with the MAM protein VDAC1 (18, 19). This association, possibly at MAM (20, 21), enables HK2, using ATP exiting mitochondria through VDAC1, to phosphorylate glucose and thereby stimulate glycolysis (22). Conversely, upon inhibition of Akt, HK2 dissociates from VDAC1, causing VDAC1 closure and increased mitochondrial membrane potential (19). This regulation of HK2 by Akt has been proposed to account for enhanced glycolysis in cancer cells, also known as the Warburg effect (23). Furthermore, Akt regulates calcium release from MAM by phosphorylating IP3R, thereby controlling apoptosis (24–26). Thus, MAM is increasingly recognized as a signaling hub controlling cell physiology (15), and is implicated in a wide spectrum of diseases, including cancer, neurodegenerative disorders, inflammation, and infection (27).The target of rapamycin (TOR) pathway is a cellular signaling cascade that, like mitochondria, is present in all eukaryotes (28, 29). TOR integrates and relays signals from both extra- and intracellular sources (e.g., growth factors, nutrients, and cellular energy levels), and thereby instructs the cell to grow. TOR is found in two structurally and functionally distinct protein complexes that in mammalian cells are termed mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2) (30). mTORC2 comprises mTOR, rictor, mammalian lethal with SEC13 protein 8 (mLST8), stress-activated protein kinase (SAPK)-interacting protein (Sin1), and protor [also known as proline-rich protein 5 (PRR5)] (31), and phosphorylates AGC kinases, such as Akt, serum/glucocorticoid-regulated kinase 1 (SGK1), and PKC, all of which are linked to cancer and diabetes (32). Growth factors activate mTORC2 by promoting mTORC2-ribosome association in a PI3K-dependent manner (33, 34). mTORC2 is antiapoptotic, presumably via its role in phosphorylating and activating Akt (34–38).Various observations indicate that mTORC2 is linked to both the ER and mitochondria. Recent findings suggest that mTORC2 is at the ER, possibly through interaction with ER-bound ribosomes (34, 39). mTORC2 phosphorylates Akt at the ER (39, 40), and mTORC2 signaling is sensitive to ER stress (41, 42). In Chlamydomonas, TOR associates with membranes from the ER (43). With regard to mitochondria, mTOR has been observed in close proximity to the outer mitochondrial membrane (44), and mTOR and mLST8 interact with the mitochondrial outer-membrane protein VDAC1 (45) and the mitochondria-associated protein Grp75 (46), respectively. mTORC2 regulates the cellular distribution of mitochondria (47), and mTORC2-activated Akt is associated with mitochondria (18, 48, 49). Pink1, a regulator of mitochondrial function, has been implicated in mTORC2 activation (50). mTORC2-addicted cancer cells exhibit enhanced dependence on mitochondria, Rab32 and HK2 (51). Finally, Barquilla et al. reported that TORC2 in trypanosomes is localized to both ER and mitochondria (52). Thus, mTORC2 has been physically and functionally linked to both the ER and mitochondria.Here we investigate the localization of mTORC2. We show that ribosome-bound mTORC2 is at MAM. Localization to MAM is growth factor-dependent. MAM-associated mTORC2 activates Akt and thereby controls MAM integrity, mitochondrial metabolism, and cell survival. Thus, our findings describe a critical role for mTORC2 in a MAM signaling hub.