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.     

An early secretory pathway mediated by GNOM-LIKE 1 and GNOM is essential for basal polarity establishment in Arabidopsis thaliana

Spatial regulation of the plant hormone indole-3-acetic acid (IAA, or auxin) is essential for plant development. Auxin gradient establishment is mediated by polarly localized auxin transporters, including PIN-FORMED (PIN) proteins. Their localization and abundance at the plasma membrane are tightly regulated by endomembrane machinery, especially the endocytic and recycling pathways mediated by the ADP ribosylation factor guanine nucleotide exchange factor (ARF-GEF) GNOM. We assessed the role of the early secretory pathway in establishing PIN1 polarity in Arabidopsis thaliana by pharmacological and genetic approaches. We identified the compound endosidin 8 (ES8), which selectively interferes with PIN1 basal polarity without altering the polarity of apical proteins. ES8 alters the auxin distribution pattern in the root and induces a strong developmental phenotype, including reduced root length. The ARF-GEF–defective mutants gnom-like 1 (gnl1-1) and gnom (van7) are significantly resistant to ES8. The compound does not affect recycling or vacuolar trafficking of PIN1 but leads to its intracellular accumulation, resulting in loss of PIN1 basal polarity at the plasma membrane. Our data confirm a role for GNOM in endoplasmic reticulum (ER)–Golgi trafficking and reveal that a GNL1/GNOM-mediated early secretory pathway selectively regulates PIN1 basal polarity establishment in a manner essential for normal plant development.Due to their sessile lifestyle, the development of plants is characterized by continuous growth, generating the capacity to adapt to environmental conditions. Such flexibility has been made possible by a set of morphological adjustments that are accomplished through altered growth regulation of different plant organs, such as leaves or roots. Most aspects of plant development are regulated by the differential distribution of the plant hormone indole-3-acetic acid (IAA, or auxin) between cells or tissues (reviewed by ref. 1). The formation of auxin maxima is generated concomitantly by local auxin biosynthesis, metabolism, and directional transport (2–8).Polar auxin transport occurs in a cell-to-cell manner and is dependent on plasma membrane-localized auxin influx and efflux carriers (reviewed by ref. 9). Among them, the PIN-FORMED (PIN) auxin efflux carriers are essential for plant development, and single or multiple pin mutants display phenotypes typical for auxin transport defects, such as tropism, embryo development, organogenesis, and root meristem patterning defects (6, 7, 10–14). A polar subcellular localization has been shown for most of the plasma membrane-localized auxin transporters, in particular for the PIN proteins (PIN1-4 and PIN7) and, to some extent, also for the ATP-BINDING CASSETTE SUBFAMILY B proteins (ABCBs) and AUXIN RESISTANT 1 (AUX1) (11–13, 15–20). The PIN proteins are known to be essential for targeting and redirecting auxin flux, which modulates the spatial pattern of expression of auxin response markers (21). PINs can be targeted toward the apical (shootward), basal (rootward), or lateral plasma membrane depending upon the PIN protein identity, the cell type, and the developmental context (reviewed by ref. 22). In the root, PIN1 is localized basally toward the root tip in stele provascular cells (12). PIN2 is also localized basally in young cortex cells close to the root meristem but is localized apically in mature cortex cells, epidermal cells, and the lateral root cap (16, 22, 23).Until now, it has been unclear whether newly synthesized PIN proteins are initially secreted to the plasma membrane in a polar or apolar manner. In Arabidopsis thaliana, the current model for PIN polar localization establishment and maintenance at the plasma membrane is based on endocytosis, polar recycling, and restriction of lateral diffusion (reviewed by ref. 24). PIN proteins are internalized via clathrin-mediated endocytosis (25, 26) and can cycle back to plasma membrane domains via distinct trafficking routes. Recycling and endocytosis of PIN1 depend on the endosome-localized fraction of the ADP ribosylation factor guanine nucleotide exchange factor (ARF-GEF) GNOM (27, 28), which is sensitive to the fungal toxin brefeldin A (BFA) (29). ARF-GEFs are essential regulators of vesicle formation and, among the eight ARF-GEFs in Arabidopsis, GNOM is the only one reported as being essential specifically for basal PIN recycling, whereas apical PIN and AUX1 localization and dynamics are not affected in gnom mutants (30, 31). Additionally, although apical targeting of AUX1 is resistant to BFA, subcellular AUX1 trafficking is BFA-sensitive, suggesting that trafficking of apical proteins may require both BFA-sensitive and -insensitive, GNOM-independent, ARF-GEF–mediated pathways (30, 32).In addition to GNOM, other Arabidopsis ARF-GEFs have been characterized, including GNOM-LIKE 1 (GNL1), which localizes to Golgi stacks and is BFA-resistant (33, 34). GNL1 acts in the early secretory pathway where it regulates COPI-mediated recycling of endoplasmic reticulum (ER)–resident proteins from the Golgi back to the ER (33, 34). Moreover, GNOM has recently been shown to predominantly localize to Golgi stacks (35) where it plays a minor but redundant function to GNL1 in ER-Golgi trafficking (33). The other Arabidopsis ARF-GEFs include GNL2, which is expressed specifically in pollen (36), and the five BIG ARF-GEFs, BIG1 to -5. BIG5, which is BFA-sensitive, has been described under the name BFA-VISUALIZED ENDOCYTIC TRAFFICKING DEFECTIVE 1 (BEN1) as mediating early endosomal trafficking (37). BIG1 to -4, of which BIG3 is BFA-resistant whereas BIG1, -2, and -4 are BFA-sensitive, have recently been described as acting redundantly in the late secretory pathway from the trans Golgi network (TGN) to the plasma membrane, as well as in late vacuolar trafficking (38).Endosomal PIN homeostasis is tightly controlled by the retromer complex through the regulation of PIN protein trafficking to the vacuole, thus controlling polar PIN abundance within the cell (39–43). Additionally, a large amount of data has demonstrated that not only trafficking routes per se are essential to determine the polar localization of PIN proteins but also internal protein signals such as posttranslational phosphorylation via the protein kinase PINOID (PID) and the protein phosphatase 2A (PP2A) (44–46). Despite recent progress, our understanding of the mechanisms establishing basal polarity remains limited. In the present work, we aimed to unravel the details of PIN basal polarity establishment by identifying selective inhibitors of this process.A number of genetic screens have been successfully used to discover new components of the endomembrane system (for examples, see refs. 34, 37, and 47–51). However, most of the molecular actors regulating endomembrane trafficking are either essential to plant survival or belong to large protein families, leading to lethality of knock-out mutants or lack of a phenotype due to redundancy. The use of fast-acting molecules suitable for the highly dynamic nature of the endomembrane system circumvents these problems and has deepened our understanding of interconnected networks of trafficking routes (52–58). While BFA has expanded our knowledge of the GNOM-dependent recycling pathway (27), other small compounds can be used to dissect different trafficking routes. In recent studies, automated screening of small molecules based on inhibition of tobacco pollen tube growth led to the isolation of a set of compounds interfering with the endomembrane system (52). Through the screening of 46,418 diverse molecules, 360 were identified as inhibitors of pollen germination (53). To dissect the trafficking routes of plasma membrane proteins specifically, a secondary screen was established based on confocal laser-scanning microscopy, leading to the identification of 123 compounds named plasma membrane recycling compound set A (PMRA), which induce mislocalization of plasma membrane markers in the Arabidopsis root meristem (53).In the present study, we reasoned that using the PMRA endomembrane trafficking modulators in combination with BFA could unravel trafficking routes regulating basal plasma membrane targeting. We designed a chemical screen to identify PMRA molecules that modulated the accumulation of PIN1 in BFA-induced agglomerations. We subsequently identified the endosidin 8 (ES8) compounds, including the original compound ES8.0 and its more potent analog ES8.1, which selectively modify PIN1 basal plasma membrane targeting in Arabidopsis with minimal effects on apical plasma membrane proteins. Using this pharmacological approach, we herein confirm that GNOM plays a role in ER-Golgi trafficking independently of its role in recycling and reveal that a GNL1/GNOM-dependent early secretory pathway is essential for targeting PIN1 toward the basal plasma membrane. Furthermore, we demonstrate that this pathway is specific for basal polarity establishment, revealing an essential and previously unknown regulatory mechanism for establishing cell polarity and regulating auxin transport and plant development.     

Cryo-EM structure of the needle filament tip complex of the Salmonella type III secretion injectisome

Type III secretion systems are multiprotein molecular machines required for the virulence of several important bacterial pathogens. The central element of these machines is the injectisome, a ∼5-Md multiprotein structure that mediates the delivery of bacterially encoded proteins into eukaryotic target cells. The injectisome is composed of a cytoplasmic sorting platform, and a membrane-embedded needle complex, which is made up of a multiring base and a needle-like filament that extends several nanometers from the bacterial surface. The needle filament is capped at its distal end by another substructure known as the tip complex, which is crucial for the translocation of effector proteins through the eukaryotic cell plasma membrane. Here we report the cryo-EM structure of the Salmonella Typhimurium needle tip complex docked onto the needle filament tip. Combined with a detailed analysis of structurally guided mutants, this study provides major insight into the assembly and function of this essential component of the type III secretion protein injection machine.

Many pathogenic or symbiotic bacteria for plants or animals have evolved specialized molecular machines known as type III protein secretion systems (T3SSs) (1–3). These machines inject bacterially encoded effector proteins into target eukaryotic cells to modulate cellular processes and ensure the survival and replication of the pathogens or symbionts that encode them (4–6). Although the structural organization of this secretion machine has been largely derived from studies of the T3SSs of the bacterial pathogens Salmonella Typhimurium and Shigella flexneri (7–15), given the conservation of its core components, it is predicted that the T3SSs in other bacteria exhibit a similar architecture. The secretion machine itself is composed of a ∼5-Md multiprotein assembly known as the injectisome (1, 7, 14, 16, 17). This core structure consists of two large substructures, an envelope-associated needle complex (7, 16, 18, 19) and a large cytoplasmic complex known as the “sorting platform” (14, 15, 20).The needle complex is composed of a multiring hollow base, which anchors the injectisome to the bacterial envelope (18). The base encloses the export apparatus, a helical structure that serves as a conduit for the passage through the inner membrane of the proteins destined to transit this secretion pathway (21). The base is also linked to a needle-like filament, which protrudes several nanometers from the bacterial surface and is formed by a single protein arranged in a helical fashion (17, 22). The needle filament, which is traversed in its entire length by a central channel ∼3 nm in diameter, is linked to the base through a structure known as the inner rod (11) and is capped at its distal end by another substructure known as the “tip complex” (23, 24).The sorting platform is located in its entirety within the cytoplasm (14, 15, 20). It is made of six pods that form a cage-like enclosure, which is capped on its cytoplasmic side by a wheel-like structure that holds a hexameric ATPase. Also harbored within the sorting platform cage is the large cytoplasmic domain of one of the components of the export apparatus, which is arranged as two concentric rings and forms a conduit for the secreted substrates to reach the entrance of the export channel (25).A distinctive feature of the T3SSs is that their activation requires contact with the target eukaryotic cell (26, 27). The activation of the T3SS is followed by the deployment of the translocon substructure, which firmly anchors the injectisome to the target cell and serves as the passageway for the effector proteins across the eukaryotic cell membrane. Although little is known about the activation process, it is thought that sensing of the target cell by the tip complex initiates a signaling event that is transduced to the secretion machine by the needle filament itself (26–31). Activation of the secretion machine is then followed by the deployment of the translocon on the target cell membrane, which along with the tip complex and the needle filament, form a continuous passageway through which effector proteins transit from the bacterial cytoplasm to the cytosol of the eukaryotic cell (23, 32, 33). The composition of the tip complex has been the subject of some controversy. While it has been proposed that in the T3SSs of Yersinia spp., Pseudomonas aeruginosa, and Salmonella spp. the tip structure is made up of a single protein, LcrV (24), PcrV (34), and SipD (35), respectively, in the case of Shigella spp., it has been alternatively proposed to be composed of two proteins, IpaB and IpaD (36), or just IpaD (37, 38). The crystal structures of monomeric SipD and close homologs show that these proteins are arranged in three domains: an N-terminal α-helical hairpin, a central coiled-coil, and a mixed α/β carboxyl-terminal domain (39–42). It has been proposed that the N-terminal α-helical hairpin domain functions as a self-chaperone that prevents the self-oligomerization and/or the premature interaction of the tip protein with the needle filament subunit within the bacterial cytoplasm (39). A current hypothesis is that during assembly at the tip of the needle, the N-terminal α-helical hairpin of SipD/IpaD is displaced to allow other domains to interact with the needle. However, there is no structural information of the fully assembled tip complex to support this hypothesis. How the tip protein assembles into the tip complex, and how it is anchored at the distal end of the needle filament, is currently unknown in large part because of the absence of a high-resolution structure of this complex. Understanding of the events that lead to the activation of the secretion machine requires detailed knowledge not only of the structure of the tip complex that caps the needle filament but, importantly, its interface with the needle filament itself.Advances in cryoelectron microscopy (cryo-EM) have allowed the visualization of most components of the T3SS machine at high resolution, both in isolation as well as in situ (7–15). However, the tip structure has eluded high-resolution visualization, in part because existing needle complex isolation protocols result in the dissociation of the tip complex from the needle filament. Here we report the visualization at high resolution by cryo-EM of the tip structure of the needle complex of the S. Typhimurium T3SS encoded within its pathogenicity island 1. Combined with functional analysis, the structure provides major insight into the potential mechanisms of injectisome assembly and activation and fills one of the remaining gaps in the quest for the high-resolution visualization of the entire T3SS injectisome.     

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.     

Class-specific interactions between Sis1 J-domain protein and Hsp70 chaperone potentiate disaggregation of misfolded proteins

Protein homeostasis is constantly being challenged with protein misfolding that leads to aggregation. Hsp70 is one of the versatile chaperones that interact with misfolded proteins and actively support their folding. Multifunctional Hsp70s are harnessed to specific roles by J-domain proteins (JDPs, also known as Hsp40s). Interaction with the J-domain of these cochaperones stimulates ATP hydrolysis in Hsp70, which stabilizes substrate binding. In eukaryotes, two classes of JDPs, Class A and Class B, engage Hsp70 in the reactivation of aggregated proteins. In most species, excluding metazoans, protein recovery also relies on an Hsp100 disaggregase. Although intensely studied, many mechanistic details of how the two JDP classes regulate protein disaggregation are still unknown. Here, we explore functional differences between the yeast Class A (Ydj1) and Class B (Sis1) JDPs at the individual stages of protein disaggregation. With real-time biochemical tools, we show that Ydj1 alone is superior to Sis1 in aggregate binding, yet it is Sis1 that recruits more Ssa1 molecules to the substrate. This advantage of Sis1 depends on its ability to bind to the EEVD motif of Hsp70, a quality specific to most of Class B JDPs. This second interaction also conditions the Hsp70-induced aggregate modification that boosts its subsequent dissolution by the Hsp104 disaggregase. Our results suggest that the Sis1-mediated chaperone assembly at the aggregate surface potentiates the entropic pulling, driven polypeptide disentanglement, while Ydj1 binding favors the refolding of the solubilized proteins. Such subspecialization of the JDPs across protein reactivation improves the robustness and efficiency of the disaggregation machinery.

Molecular chaperones are involved in the maintenance of protein homeostasis by aiding correct protein folding (1). Yet severe stress conditions induce excessive protein misfolding and aggregation (2). Upon stress relief, the return to the proteostasis is mediated by the Hsp70 chaperone with cochaperones, including J-domain proteins (JDPs/Hsp40s), which together restore the native state of misfolded polypeptides trapped in aggregates (3–5). The JDP–Hsp70 system acts alone in metazoans or in cooperation with an Hsp100 disaggregase in most other eukaryotes and bacteria (5, 6).Protein disaggregation and refolding starts with a recognition of misfolded polypeptides within an aggregate by a JDP, and then, its J-domain interacts with the nucleotide-binding domain of Hsp70, inducing ATP hydrolysis which triggers the closure of the Hsp70’s substrate-binding domain over the aggregated substrate (7, 8). The aggregate-bound Hsp70 interacts with an Hsp100 disaggregase, and this interaction allosterically activates Hsp100 and tethers it to the aggregate (9–16). Subsequently, in an ATP-driven process, Hsp100 disentangles and translocates polypeptides from aggregates (17–21), which enables their correct refolding, spontaneous or with an assistance of Hsp70 and its cochaperones (22, 23).JDPs are the major regulators of the Hsp70 activity and substrate specificity (3, 24, 25). In yeast Saccharomyces cerevisiae, a general Hsp70 chaperone, Ssa1, is recruited to protein disaggregation by two main cytosolic JDPs, Ydj1 and Sis1, assigned to the Class A and Class B, respectively (3, 4, 26). Both Ydj1 and Sis1 comprise a helical, highly conserved J-domain, a flexible, mostly unstructured G/F region, two beta-barrel peptide-binding domains, CTDI and CTDII, and a C-terminal dimerization domain (27–33). Ydj1 additionally features a Zn-binding domain located in the first part of the CTDI region of the protein, which is distinctive for the Class A JDPs (32, 34).Despite the structural similarities, the two JDPs are functionally nonredundant. Sis1 is essential, and Ydj1 is required for growth above 34 °C (26, 27, 35, 36). Overexpression of Sis1 suppresses the phenotype caused by the deletion of YDJ1, while Ydj1 overexpression is not sufficient to suppress the deletion of SIS1 (26, 27, 35–37). The two JDPs show different specificities toward amorphous and amyloid aggregates (35, 38) and different populations of amorphous aggregates formed in vitro (4, 24).Recent reports shed more light on the JDPs’ divergence. Both JDPs form homodimers, which differ in the structural orientation of the J-domain: In Sis1, the J-domain is restrained from Hsp70 binding by the interaction with the Helix 5 in the G/F region (26, 33, 39–41). Such autoinhibition, which also occurs in most human Class B JDPs, is released through the interaction with the C-terminal EEVD motif of Hsp70 (33, 42). This regulation is important for the disassembly of amyloid fibrils by the human JDP–Hsp70 system (43), but its role in the handling of stress-related, amorphous aggregates is not clear. Despite the breadth of data on Hsp70 mechanisms, we still lack understanding of how the disparate features of the JDPs impact Hsp70 functioning in protein disaggregation.Here, we investigate individual steps of protein disaggregation in the context of functional differences between Sis1 and Ydj1. Using various biochemical approaches, we show that the two JDPs drive different modes of Ssa1 binding to aggregated substrates, which dictate diverse kinetics of their disaggregation by Hsp104. The distinctive performance of Sis1 is associated with its interaction with the C terminus of Hsp70. Our results suggest that the bivalent interaction with the Class B JDP conditions aggregate remodeling by the Hsp70 system, resulting in enhanced Hsp104-dependent protein recovery. Our data indicate a mechanism by which the Class A and B JDPs contribute to the disaggregation efficacy in a complex and divergent manner.     

Mutant libraries reveal negative design shielding proteins from supramolecular self-assembly and relocalization in cells

Understanding the molecular consequences of mutations in proteins is essential to map genotypes to phenotypes and interpret the increasing wealth of genomic data. While mutations are known to disrupt protein structure and function, their potential to create new structures and localization phenotypes has not yet been mapped to a sequence space. To map this relationship, we employed two homo-oligomeric protein complexes in which the internal symmetry exacerbates the impact of mutations. We mutagenized three surface residues of each complex and monitored the mutations’ effect on localization and assembly phenotypes in yeast cells. While surface mutations are classically viewed as benign, our analysis of several hundred mutants revealed they often trigger three main phenotypes in these proteins: nuclear localization, the formation of puncta, and fibers. Strikingly, more than 50% of random mutants induced one of these phenotypes in both complexes. Analyzing the mutant’s sequences showed that surface stickiness and net charge are two key physicochemical properties associated with these changes. In one complex, more than 60% of mutants self-assembled into fibers. Such a high frequency is explained by negative design: charged residues shield the complex from self-interacting with copies of itself, and the sole removal of the charges induces its supramolecular self-assembly. A subsequent analysis of several other complexes targeted with alanine mutations suggested that such negative design is common. These results highlight that minimal perturbations in protein surfaces’ physicochemical properties can frequently drive assembly and localization changes in a cellular context.

Understanding genotype to phenotype relationships is crucial to predict the molecular consequences of mutations (1). At the protein level, alanine scans have revealed how individual residues contribute to protein function, stability, and binding affinity (2–4). More recently, systematic mappings have been widely used to connect sequence variability to changes in protein structure (5, 6), stability (7–9), solubility (10), and functionality (2, 11–14). Similar efforts have been made to map the impact of mutations in protein–ligand (15, 16) and protein–protein interactions (17–21).However, mutations can impact proteins beyond their stability, function, or existing interactions with specific partners or ligands. Sequences can also encode how proteins distribute spatially in cells, either by addressing them to membrane-bound compartments (22) or by inducing their self-assembly into large polymeric structures (23–27) and membraneless compartments (28, 29). While changes in protein self-assembly and localization can serve a functional purpose in adaptation (30–36), they can also lead to disease (37). For example, the supramolecular self-assembly of hemoglobin and γD-crystallin cause sickle-cell disease and cataracts, respectively (38, 39). The mislocalization of nuclear proteins TDP-43 and FUS in the cytosol is associated with amyotrophic lateral sclerosis disease (40, 41), and the mislocalization of Ataxin-3 to the nucleus has been implicated in spinocerebellar ataxia type 3 disease (42). It is therefore critical to characterize principles by which mutations can trigger such supramolecular self-assembly and mislocalization.Symmetry is frequent in proteins (37, 43) and is a crucial property promoting their self-assembly into high-order structures (44–50). Indeed, a strong enrichment in symmetric homo-oligomers among natural filament-forming proteins has been reported (37). Previous work has also shown that point mutations to two hydrophobic amino acids—leucine and tyrosine—frequently led symmetric homo-oligomers to assemble into high-order assemblies. However, whether other types of amino acids would display a similar potential, whether they would do so often, and whether additional phenotypes of assembly and localization could emerge upon mutation remains unknown.Here, we assess the potential of mutations to trigger such changes in protein assembly and localization in vivo. We targeted two homo-oligomeric protein complexes and randomly mutated three neighboring residues at the surface of each complex. We expressed the mutants fused to a fluorescent protein to track their spatial distribution in yeast cells. We found that a vast sequence space led to changes in protein assembly and localization in both proteins with three predominant phenotypes: nuclear localization, the formation of filaments, and the formation of puncta. Sequencing of the mutants revealed that increasing surface stickiness frequently promoted nuclear localization in one of the two proteins. Surprisingly, in the other protein, a loss of negatively charged residues was sufficient to trigger protein self-assembly, with fibers frequently forming regardless of the type of mutation, including to alanine and glycine. We also observed that four out of eight additional complexes analyzed underwent supramolecular self-assembly or a change in cellular localization when surface charges were mutated to alanine, implying that negative design against supramolecular self-assembly and mislocalization is common among symmetric homo-oligomers.     

Retro-translocation of mitochondrial intermembrane space proteins

The content of mitochondrial proteome is maintained through two highly dynamic processes, the influx of newly synthesized proteins from the cytosol and the protein degradation. Mitochondrial proteins are targeted to the intermembrane space by the mitochondrial intermembrane space assembly pathway that couples their import and oxidative folding. The folding trap was proposed to be a driving mechanism for the mitochondrial accumulation of these proteins. Whether the reverse movement of unfolded proteins to the cytosol occurs across the intact outer membrane is unknown. We found that reduced, conformationally destabilized proteins are released from mitochondria in a size-limited manner. We identified the general import pore protein Tom40 as an escape gate. We propose that the mitochondrial proteome is not only regulated by the import and degradation of proteins but also by their retro-translocation to the external cytosolic location. Thus, protein release is a mechanism that contributes to the mitochondrial proteome surveillance.Mitochondrial biogenesis is essential for eukaryotic cells. Because most mitochondrial proteins originate in the cytosol, mitochondria had to develop a protein import system. Given the complex architecture of these organelles, with two membranes and two aqueous compartments, protein import and sorting require the cooperation of several pathways. The main entry gate for precursor proteins is the translocase of the outer mitochondrial membrane (TOM) complex. Upon entering mitochondria, proteins are routed to different sorting machineries (1–5).Reaching the final location is one step in the maturation of mitochondrial proteins that must be accompanied by their proper folding. The mitochondrial intermembrane space assembly (MIA) pathway for intermembrane space (IMS) proteins illustrates the importance of coupling these processes because this pathway links protein import with oxidative folding (6–10). Upon protein synthesis in the cytosol, the cysteine residues of IMS proteins remain in a reduced state, owing to the reducing properties of the cytosolic environment (11, 12). After entering the TOM channel, precursor proteins are specifically recognized by Mia40 protein, and their cysteine residues are oxidized through the cooperative action of Mia40 and Erv1 proteins (7, 13–17). Mia40 is a receptor, folding catalyst, and disulfide carrier, and the Erv1 protein serves as a sulfhydryl oxidase. The oxidative folding is believed to provide a trapping mechanism that prevents the escape of proteins from the IMS back to the cytosol (10, 13, 18). Our initial result raised a possibility that the reverse process can also occur, as we observed the relocation of in vitro imported Tim8 from mitochondria to the incubation buffer (13). Thus, we sought to establish whether and how this process can proceed in the presence of the intact outer membrane (OM). Our study provides, to our knowledge, the first characterization of the mitochondrial protein retro-translocation. The protein retro-translocation serves as a regulatory and quality control mechanism for the mitochondrial IMS proteome.     

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).     

Differential gradients of interaction affinities drive efficient targeting and recycling in the GET pathway

Efficient and accurate localization of membrane proteins requires a complex cascade of interactions between protein machineries. This requirement is exemplified in the guided entry of tail-anchored (TA) protein (GET) pathway, where the central targeting factor Get3 must sequentially interact with three distinct binding partners to ensure the delivery of TA proteins to the endoplasmic reticulum (ER) membrane. To understand the molecular principles that provide the vectorial driving force of these interactions, we developed quantitative fluorescence assays to monitor Get3–effector interactions at each stage of targeting. We show that nucleotide and substrate generate differential gradients of interaction energies that drive the ordered interaction of Get3 with successive effectors. These data also provide more molecular details on how the targeting complex is captured and disassembled by the ER receptor and reveal a previously unidentified role for Get4/5 in recycling Get3 from the ER membrane at the end of the targeting reaction. These results provide general insights into how complex protein interaction cascades are coupled to energy inputs in biological systems.Membrane proteins comprise ∼30% of the proteome; their efficient and accurate localization is crucial for the structure and function of all cells. Although the well-studied cotranslational signal recognition particle pathway delivers numerous endoplasmic reticulum (ER) -destined proteins (1), many membrane proteins use posttranslational targeting pathways with mechanisms that are far less well understood. A salient example is tail-anchored (TA) proteins, which comprise 3–5% of the eukaryotic membrane proteome and play essential roles in numerous processes, including protein translocation, vesicular trafficking, quality control, and apoptosis (2–5). Because their sole transmembrane domain is at the extreme C terminus, TA proteins cannot engage the cotranslational signal recognition particle machinery and instead, must use posttranslational pathways for localization (6).In the guided entry of TA protein (GET) pathway, TA proteins are initially captured by the yeast cochaperone Sgt2 (or mammalian SGTA) (2, 7). The Get4/5 complex then enables loading of the TA substrate from Sgt2 onto Get3 (or mammalian TRC40), the central targeting factor (7–9). The Get3/TA complex binds a receptor complex on the ER membrane comprised of Get1 and Get2, through which the TA protein is released from Get3 and inserted into the membrane (10–12). Dissociation from Get1/2 is then needed to recycle Get3 for additional rounds of targeting (11–13). Knockout of Get3 (or TRC40) confers stress sensitivity in yeast and embryonic lethality in mammals, underscoring its essential role in the proper functioning of the cell (10, 14, 15).TA protein targeting is driven by the ATPase cycle of Get3, a member of the signal recognition particle, MinD and BioD class of nucleotide hydrolases (8, 16). Crystallographic studies revealed that Get3 is an obligate homodimer, in which the ATPase domains bridge the dimer interface and are connected to helical domains (17, 18). Notably, the conformation of Get3 can be tuned by its nucleotide state, the TA substrate, and its binding partners (11, 12, 17, 19). Apo-Get3 is in an open conformation, in which the helical domains are disconnected (18). ATP biases Get3 to more closed structures, in which the helical domains form a contiguous hydrophobic surface implicated in TA protein binding (17, 18, 20). The Get4/5 complex further locks Get3 into an occluded conformation, in which ATP is tightly bound, but its hydrolysis is delayed, priming Get3 into the optimal state to capture the TA substrate (19, 21). TA proteins induce additional association of Get3 dimers to form a closed tetramer, which stimulates rapid ATP hydrolysis and delays ADP release (19, 22). Finally, Get1 strongly binds apo-Get3 in the open conformation (see below), likely at the end of the targeting reaction (11, 12, 23).The GET pathway demands a sequential cascade of interactions of Get3 with three distinct binding partners: the Get4 subunit in the Get4/5 complex and the Get1 and Get2 subunits in the Get1/2 receptor complex. All three partners share overlapping binding sites on Get3 (Fig. S1) (21), raising intriguing questions as to the mechanisms that ensure the high spatial and temporal accuracy of these protein interactions. For example, Get3 must first interact with Get4/5 in the cytosol to facilitate the loading of TA substrate (7, 9). It is unclear what then drives the release of Get3 from Get4/5 and enables its transit to the ER membrane, where it interacts with the Get1/2 receptor instead.Similarly, how Get3 and the Get3/TA complex transit between different subunits of the Get1/2 receptor at the ER membrane remains unclear. Get1/2 (WRB/CAML in mammals) is necessary and sufficient for TA protein insertion at the ER membrane (12, 13, 24, 25). Crystallographic analyses revealed that Get1 binds strongly to apo- or ADP-bound Get3 in the open conformation (11, 12, 23), whereas Get2 can bind Get3 in semiclosed or closed states (11, 12). In vitro reconstitution experiments showed that high concentrations of Get1 but not Get2 can trigger substrate release from Get3 (12). These observations led to the model that Get2 first captures Get3, whereas Get1 is responsible for disassembling the targeting complex (2, 13). Nevertheless, the subunit that is responsible for capturing the Get3/TA targeting complex has not been experimentally addressed, and whether Get1 or Get2 can discriminate different substrate-bound states of Get3 also has not been addressed.At the end of targeting, Get1 is tightly bound to apo-Get3 (11–13). Experiments with the cytosolic domain (CD) of Get1 show that its interaction with Get3 is strongly antagonized by ATP, leading to the current model that ATP drives the recycling of Get3 from the ER membrane (11, 12). However, two observations raise difficulties with this minimal model. In experiments with intact ER membranes or Get1/2 proteoliposomes (PLs), ATP is insufficient to completely release Get3 from the membrane (12, 13). Furthermore, the tight interaction of Get1 with Get3 raises the possibility that their dissociation is slow (11), which could pose potential barriers for subsequent rounds of TA protein targeting.To address these issues, we developed fluorescence assays to report on the interaction of Get3 with its effectors. Quantitative measurements show that both substrate and nucleotide regulate the interaction of Get3 with Get4/5 and Get1/2, generating differential gradients of interaction energies that drive the ordered transit of Get3 from one binding partner to the next. These results also reveal an active role of ATP in displacing Get3 from Get1, which together with Get4/5, ensures the effective recycling of Get3 back to the cytosol.     

Phosphatidylinositol-4-phosphate-dependent membrane traffic is critical for fungal filamentous growth

The phospholipid phosphatidylinositol-4-phosphate [PI(4)P], generated at the Golgi and plasma membrane, has been implicated in many processes, including membrane traffic, yet its role in cell morphology changes, such as the budding to filamentous growth transition, is unknown. We show that Golgi PI(4)P is required for such a transition in the human pathogenic fungus Candida albicans. Quantitative analyses of membrane traffic revealed that PI(4)P is required for late Golgi and secretory vesicle dynamics and targeting and, as a result, is important for the distribution of a multidrug transporter and hence sensitivity to antifungal drugs. We also observed that plasma membrane PI(4)P, which we show is functionally distinct from Golgi PI(4)P, forms a steep gradient concomitant with filamentous growth, despite uniform plasma membrane PI-4-kinase distribution. Mathematical modeling indicates that local PI(4)P generation and hydrolysis by phosphatases are crucial for this gradient. We conclude that PI(4)P-regulated membrane dynamics are critical for morphology changes.Phosphatidylinositol-4-phosphate [PI(4)P] is a minor constituent of cellular membranes that is essential for polarized growth, membrane traffic, and cytoskeleton organization (1–3). The majority of PI(4)P in budding yeast is generated by two essential PI-4-kinases, Pik1 at the Golgi and Stt4 at the plasma membrane (PM) (4–7). Although we have shown that PM Stt4 and the PI(4)P-5-kinase Mss4 are critical for the human fungal pathogen Candida albicans filamentous growth (8), little is known regarding the importance of Golgi PI(4)P. Perturbation of Golgi PI(4)P levels in Saccharomyces cerevisiae and mammalian cells results in defects in Golgi morphology and secretion (9–13). Furthermore, the Golgi in mammalian cells is important for cell polarity (14). In the filamentous fungus Neurospora crassa, PI(4)P has been observed at the Golgi (15), yet its function is unknown.In a range of fungi, including pathogenic species, a morphological transition between yeast and filamentous forms, triggered by numerous external stimuli, is important for virulence (16, 17). Many proteins localize to the tip of the C. albicans protruding filament and a number of proteins are either secreted or incorporated into the cell wall during the yeast to filamentous morphological transition (17), alluding to the importance of membrane traffic in this process. Here we show that cells with reduced Golgi PI(4)P levels are defective in morphogenesis and that Golgi PI(4)P is critical for two distinct steps in the secretory pathway. Furthermore, we observed a striking gradient of PM PI(4)P along the length of the hyphal filament and mathematical modeling revealed the processes crucial for this distribution.     

Membrane adhesion dictates Golgi stacking and cisternal morphology

Two classes of proteins that bind to each other and to Golgi membranes have been implicated in the adhesion of Golgi cisternae to each other to form their characteristic stacks: Golgi reassembly and stacking proteins 55 and 65 (GRASP55 and GRASP65) and Golgin of 45 kDa and Golgi matrix protein of 130 kDa. We report here that efficient stacking occurs in the absence of GRASP65/55 when either Golgin is overexpressed, as judged by quantitative electron microscopy. The Golgi stacks in these GRASP-deficient HeLa cells were normal both in morphology and in anterograde cargo transport. This suggests the simple hypothesis that the total amount of adhesive energy gluing cisternae dictates Golgi cisternal stacking, irrespective of which molecules mediate the adhesive process. In support of this hypothesis, we show that adding artificial adhesive energy between cisternae and mitochondria by dimerizing rapamycin-binding domain and FK506-binding protein domains that are attached to cisternal adhesive proteins allows mitochondria to invade the stack and even replace Golgi cisternae within a few hours. These results indicate that although Golgi stacking is a highly complicated process involving a large number of adhesive and regulatory proteins, the overriding principle of a Golgi stack assembly is likely to be quite simple. From this simplified perspective, we propose a model, based on cisternal adhesion and cisternal maturation as the two core principles, illustrating how the most ancient form of Golgi stacking might have occurred using only weak cisternal adhesive processes because of the differential between the rate of influx and outflux of membrane transport through the Golgi.The Golgi apparatus plays a central role in the processing, sorting, and secretion of various cargo molecules destined for various intracellular and extracellular destinations (1). In animal and plant cells, its unique structure of four to six stacked, roughly planar cisternae serves, among other things, as a platform to organize Golgi resident glycosyltransferases into distinct membrane-bound subcompartments (the cis-, medial-, and trans-Golgi cisternae) for proper and sequential posttranslational maturation of the transiting cargo proteins (2, 3).Although these characteristic features of Golgi morphology have drawn the attention of many researchers for many decades, the molecular mechanisms underlying them are still unclear (4). Pioneering functional reconstitution studies using a cell-free system in which Golgi stacks (but not ribbons) reassemble from mitotic extracts (5–8) yielded two classes of purified proteins, each clearly contributing to stacking: globular Golgi reassembly and stacking proteins (GRASPs; the homologous proteins GRASP65 and GRASP55) (7, 9) and the helical rod-like and partially homologous proteins Golgi matrix protein of 130 kDa (GM130) and Golgin of 45 kDa (Golgin45) (10, 11). One member of each family (GRASP65 and GM130) is located in the cis-most cisterna (7, 12), and another member of each family (GRASP55 and Golgin45) is located in this (and more so in later cisternae) (9, 10). The GRASP proteins bind to the Golgins (note that we use the term “Golgin” in a limited sense in this article to refer either to GM130 or Golgin45) (10, 11). An appealing mechanism for intercisternal adhesion has been proposed for the GRASP proteins based on X-ray crystallography and biochemistry that involves PSD-95, Dlg1, Zo-1 domain-dependent homo-oligomerization in trans (13–15). In recent years, with the advent of RNAi-based technologies, knock-down studies have broadly confirmed a role for GRASP proteins and Golgins in controlling Golgi morphology but have not agreed with each other on many notable details, leaving the field in a somewhat confused and conflictory state (10, 15–20). In the simpler case of Drosophila, dsRNA-mediated depletion of dGRASP results in ∼30% loss of Golgi stacks, whereas double depletion of dGRASP and dGM130 was shown to unstack the Golgi, as examined by EM (21). In Schizosaccharomyces pombe, depletion of yeast GRASP homolog Grh1 has no effect on Golgi stacking (22). This situation can be extended to plants, where no GRASP or even Golgin homolog has been identified thus far (23).To address this discrepancy, we studied the relative contribution of these four “stacking factors” (GM130, Golgin45, GRASP65, and GRASP55) for Golgi stacking by extensive quantitative analysis of EM-based studies. The results of these experiments strongly indicate that the overriding principle of Golgi stack assembly is simple cisternal adhesion, regardless of which molecule mediates the cisternal adhesive process. We show that adhesive energy that binds cisternae to each other at physiological equilibrium can be generated by many different combinations of Golgins+GRASPs or even in the absence of GRASPs. On the basis of this new understanding, we propose a simple mechanistic model illustrating how the most ancient form of Golgi stacking might have been facilitated by the principles described in our adhesion model and cisternal maturation (i.e., Rab conversion) in organisms, such as yeast (S. pombe) and plants, for which these four adhesive proteins either are not found or are not important for stacking.     

ER trapping reveals Golgi enzymes continually revisit the ER through a recycling pathway that controls Golgi organization

Whether Golgi enzymes remain localized within the Golgi or constitutively cycle through the endoplasmic reticulum (ER) is unclear, yet is important for understanding Golgi dependence on the ER. Here, we demonstrate that the previously reported inefficient ER trapping of Golgi enzymes in a rapamycin-based assay results from an artifact involving an endogenous ER-localized 13-kD FK506 binding protein (FKBP13) competing with the FKBP12-tagged Golgi enzyme for binding to an FKBP-rapamycin binding domain (FRB)-tagged ER trap. When we express an FKBP12-tagged ER trap and FRB-tagged Golgi enzymes, conditions precluding such competition, the Golgi enzymes completely redistribute to the ER upon rapamycin treatment. A photoactivatable FRB-Golgi enzyme, highlighted only in the Golgi, likewise redistributes to the ER. These data establish Golgi enzymes constitutively cycle through the ER. Using our trapping scheme, we identify roles of rab6a and calcium-independent phospholipase A₂ (iPLA₂) in Golgi enzyme recycling, and show that retrograde transport of Golgi membrane underlies Golgi dispersal during microtubule depolymerization and mitosis.The Golgi apparatus is the major processing and sorting station at the crossroads of the secretory pathway (1–4). It receives newly synthesized proteins from the endoplasmic reticulum (ER), processes them using Golgi-specific enzymes, and sorts them to the plasma membrane (PM) and other final destinations. During this process, specialized sorting and transport machinery of the Golgi filter out selected membrane and protein components, returning them back to the ER for continued use. How the Golgi maintains its structure and function amid this ongoing bidirectional membrane trafficking has been a long-standing debate.A variety of studies have advanced the view that the Golgi apparatus is a dynamic, steady-state system in which resident enzymes continuously recycle back to the ER and return to the Golgi through the same retrograde and anterograde trafficking routes used by other proteins (4–6). This model helps account for the striking dispersal of the Golgi when ER export is blocked (7–9), microtubules are depolymerized (10–12), or cells enter mitosis (13, 14). In each case, Golgi enzymes have been reported to redistribute to the site of transport block in the ER.An alternative model has been proposed that envisions the Golgi as an autonomous organelle with stable components that provide a template for its growth and division. In this model, Golgi enzymes ordinarily remain localized in the Golgi throughout their lifetime and Golgi dispersal under ER export blockade, microtubule depolymerization, or mitotic entry is due to a reversible breakdown of the Golgi itself into smaller elements or vesicles, without contact with the ER or its associated pathways. One primary support for this model comes from studies examining the relationship of Golgi enzymes and ER proteins using a rapamycin-induced protein heterodimerization assay to trap FKBP12- and FKBP-rapamycin binding domain (FRB)-tagged probes in close proximity (15). Using an FRB-tagged, ER-retained version of invariant chain Iip35 (Ii-FRB) (16, 17) and FKBP12-labeled Golgi enzymes, these studies observed little trapping of Golgi enzymes in the ER in the presence of rapamycin (15), including during Golgi dispersal upon mitotic entry (18).If representative of normal Golgi enzyme behavior, the ER trapping assay’s results would call into question the wide range of other observations supporting the steady-state Golgi model and its ER dependency. We thus examined the assay in more detail to determine whether it might be underestimating the extent of Golgi enzyme recycling to the ER. Prior work has shown that an endogenous FK506-binding protein, FKBP13, localizes to the lumen of the ER (19, 20), where, in the presence of rapamycin, it forms a ternary complex with FRB-containing proteins (20). We tested the possibility that binding of endogenous FKBP13 to ER-localized Ii-FRB when rapamycin is introduced might explain why no significant trapping of FKBP12-labeled Golgi enzymes in the ER was observed in the prior work. Consistent with this possibility, we found that coexpressing an FKBP12-tagged ER protein and an FRB-tagged Golgi enzyme marker, conditions where endogenous ER-localized FKBP13 would not sequester the ER trap, resulted in complete redistribution of the Golgi marker into the ER within 4 h of rapamycin treatment. In the ER, FRB-Golgi and FKBP12-ER markers underwent FRET, indicating direct binding upon rapamycin-induced redistribution. These data support the Golgi recycling theory by providing evidence of trapping of Golgi enzymes in the ER.The ability to assess retrograde transport of Golgi enzymes using our modified ER trapping assay enabled us to characterize the pathway while offering additional evidence of its existence. We found that the carriers delivering Golgi enzymes to the ER were tubule-shaped and moved peripherally after extending off from the Golgi. Rab6a and cation-independent phospholipase A₂ (iPLA₂) were required for their delivery to the ER. Golgi enzyme recycling to the ER was also shown to be involved in Golgi fragmentation during microtubule depolymerization and during mitosis. We anticipate that further use of this modified ER trapping assay will provide more insights into the precise nature of the Golgi’s dependence on the ER.     

Conformational dynamics of a crystalline protein from microsecond-scale molecular dynamics simulations and diffuse X-ray scattering

X-ray diffraction from protein crystals includes both sharply peaked Bragg reflections and diffuse intensity between the peaks. The information in Bragg scattering is limited to what is available in the mean electron density. The diffuse scattering arises from correlations in the electron density variations and therefore contains information about collective motions in proteins. Previous studies using molecular-dynamics (MD) simulations to model diffuse scattering have been hindered by insufficient sampling of the conformational ensemble. To overcome this issue, we have performed a 1.1-μs MD simulation of crystalline staphylococcal nuclease, providing 100-fold more sampling than previous studies. This simulation enables reproducible calculations of the diffuse intensity and predicts functionally important motions, including transitions among at least eight metastable states with different active-site geometries. The total diffuse intensity calculated using the MD model is highly correlated with the experimental data. In particular, there is excellent agreement for the isotropic component of the diffuse intensity, and substantial but weaker agreement for the anisotropic component. Decomposition of the MD model into protein and solvent components indicates that protein–solvent interactions contribute substantially to the overall diffuse intensity. We conclude that diffuse scattering can be used to validate predictions from MD simulations and can provide information to improve MD models of protein motions.Proteins explore many conformations while carrying out their functions in biological systems (1–3). X-ray crystallography is the dominant source of information about protein structure; however, crystal structure models usually consist of just a single major conformation and at most a small portion of the model as alternate conformations. Crystal structures therefore are missing many details about the underlying conformational ensemble (4).Proteins assembled in crystalline arrays, like proteins in solution, exhibit rich conformational diversity (4) and often can perform their native functions (5). Many methods have emerged for using Bragg data to model conformational diversity in protein crystals (6–17). The development of these methods has been important as conformational diversity can lead to inaccuracies in protein structure models (9, 18–20). A key limitation of using the Bragg data, however, is that different models of conformational diversity can yield the same mean electron density.Whereas the Bragg scattering only contains information about the mean electron density, diffuse scattering (diffraction resulting in intensity between the Bragg peaks) is sensitive to spatial correlations in electron density variations (21–28) and therefore contains information about the way that atomic positions vary together in protein crystals. Because models that yield the same mean electron density can yield different correlations in electron density variations, diffuse scattering provides a means to increase the accuracy of crystallography for determining protein conformational variations (29). Peter Moore (30) and Mark Wilson (31) have argued that diffuse scattering should be used to test models of conformational diversity in X-ray crystallography.Several pioneering studies used diffuse scattering to reveal insights into correlated motions in proteins (17, 30, 32–49). Some of these studies used diffuse scattering to experimentally validate predictions of correlated motions from molecular-dynamics (MD) simulations (35–37, 40, 42–44). These studies revealed important insights but were limited by inadequate sampling of the conformational ensemble, leading to lack of convergence of the diffuse scattering calculations (35). Microsecond-scale simulations of staphylococcal nuclease were predicted to be adequate for convergence of diffuse scattering calculations (42). Modern simulation algorithms and computer hardware now enable microsecond or longer MD simulations of protein crystals (50).Here, we present calculations of diffuse X-ray scattering using a 1.1-μs MD simulation of crystalline staphylococcal nuclease. The results demonstrate that we have overcome the past limitation of inadequate sampling. We chose staphylococcal nuclease because the experiments of Wall et al. (49) still represent the only complete, high-quality, 3D diffuse scattering data set from a protein crystal. The calculated diffuse intensity is very similar using two independent halves of the trajectory; the results therefore are reproducible and can be meaningfully compared with the experimental data. The MD simulation provides a rich picture of conformational diversity in the energy landscape of a protein crystal, consisting of at least eight metastable states. Like previous MD studies of crystalline staphylococcal nuclease (42–44), the agreement of the simulation with the total experimental diffuse intensity is excellent, supporting the use of MD simulations to model diffuse scattering data. Unlike previous MD studies, we separately compared the more finely structured, anisotropic component of the diffuse intensity with experimental data. The agreement is substantial but weaker than for the isotropic component, indicating there are inaccuracies in the MD models. Our results therefore point toward using diffuse scattering to improve MD models of protein motions.     

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.     

Visualization of the type III secretion sorting platform of Shigella flexneri

Bacterial type III secretion machines are widely used to inject virulence proteins into eukaryotic host cells. These secretion machines are evolutionarily related to bacterial flagella and consist of a large cytoplasmic complex, a transmembrane basal body, and an extracellular needle. The cytoplasmic complex forms a sorting platform essential for effector selection and needle assembly, but it remains largely uncharacterized. Here we use high-throughput cryoelectron tomography (cryo-ET) to visualize intact machines in a virulent Shigella flexneri strain genetically modified to produce minicells capable of interaction with host cells. A high-resolution in situ structure of the intact machine determined by subtomogram averaging reveals the cytoplasmic sorting platform, which consists of a central hub and six spokes, with a pod-like structure at the terminus of each spoke. Molecular modeling of wild-type and mutant machines allowed us to propose a model of the sorting platform in which the hub consists mainly of a hexamer of the Spa47 ATPase, whereas the MxiN protein comprises the spokes and the Spa33 protein forms the pods. Multiple contacts among those components are essential to align the Spa47 ATPase with the central channel of the MxiA protein export gate to form a unique nanomachine. The molecular architecture of the Shigella type III secretion machine and its sorting platform provide the structural foundation for further dissecting the mechanisms underlying type III secretion and pathogenesis and also highlight the major structural distinctions from bacterial flagella.Type III secretion systems (T3SSs) are essential virulence determinants for many Gram-negative pathogens. The injectisome, also known as the needle complex, is the central T3SS machine required to inject effector proteins from the bacterium into eukaryotic host cells (1, 2). The injectisome has three major components: an extracellular needle, a basal body, and a cytoplasmic complex (3). Contact with a host cell membrane triggers activation of the injectisome and the insertion of a translocon pore into the target cell membrane. The entire complex then serves as a conduit for direct translocation of effectors (1, 2). Assembly of a functional T3SS requires recognition and sorting of specific secretion substrates in a well-defined order by the cytoplasmic complex (4, 5). Furthermore, genes encoding the cytoplasmic complex are regulated by physical and environmental signals (6), providing temporal control of the injection of effector proteins and thereby optimizing invasion and virulence.Significant progress has been made in elucidating T3SS structures from many different bacteria (7, 8). 3D reconstructions of purified injectisomes from Salmonella and Shigella, together with the atomic structures of major basal body proteins, have provided a detailed view of basal body architecture (9, 10). Recent in situ structures of injectisomes from Shigella flexneri, Salmonella enterica, and Yersinia enterocolitica revealed an export gate and the structural flexibility of the basal body (11, 12). Unfortunately, these in situ structures from intact bacteria (11, 12) did not reveal any evident densities related to the proposed model of the cytoplasmic complex (8, 13).The flagellar C ring is the cytoplasmic complex in evolutionarily related flagellar systems. It is composed of flagellar proteins FliG, FliM, and FliN and plays an essential role in flagellar assembly, rotation, and switching (14). Large drum-shaped structures of the flagellar C ring have been determined in both purified basal bodies (15, 16) and in situ motors (17–19). Similarly, electron microscopy analysis in Shigella indicated that the Spa33 protein (a homolog of the flagellar proteins FliN and FliM) is localized beneath the basal body via interactions with MxiG and MxiJ and is an essential component of the putative C ring (20). Recent experimental evidence suggests that the putative C ring provides a sorting platform for the recognition and secretion of the substrates in S. enterica (5). This sorting platform consists of three proteins, SpaO, OrgA, and OrgB, which are highly conserved among other T3SSs (21) (SI Appendix, Table S1). Despite its critical roles, little is still known about the structure and assembly of the cytoplasmic sorting platform in T3SS. In this study, we choose S. flexneri as a model system to study the intact T3SS machine and its cytoplasmic complex, mainly because a wealth of structural, biochemical, and functional information is available for the S. flexneri T3SS (22).