Yeast homologues of tomosyn and lethal giant larvae function in exocytosis and are associated with the plasma membrane SNARE, Sec9.

We have identified a pair of related yeast proteins, Sro7p and Sro77p, based on their ability to bind to the plasma membrane SNARE (SNARE) protein, Sec9p. These proteins show significant similarity to the Drosophila tumor suppressor, lethal giant larvae and to the neuronal syntaxin-binding protein, tomosyn. SRO7 and SRO77 have redundant functions as loss of both gene products leads to a severe cold-sensitive growth defect that correlates with a severe defect in exocytosis. We show that similar to Sec9, Sro7/77 functions in the docking and fusion of post-Golgi vesicles with the plasma membrane. In contrast to a previous report, we see no defect in actin polarity under conditions where we see a dramatic effect on secretion. This demonstrates that the primary function of Sro7/77, and likely all members of the lethal giant larvae family, is in exocytosis rather than in regulating the actin cytoskeleton. Analysis of the association of Sro7p and Sec9p demonstrates that Sro7p directly interacts with Sec9p both in the cytosol and in the plasma membrane and can associate with Sec9p in the context of a SNAP receptor complex. Genetic analysis suggests that Sro7 and Sec9 function together in a pathway downstream of the Rho3 GTPase. Taken together, our studies suggest that members of the lethal giant larvae/tomosyn/Sro7 family play an important role in polarized exocytosis by regulating SNARE function on the plasma membrane.     

The yeast lgl family member Sro7p is an effector of the secretory Rab GTPase Sec4p

Rab guanosine triphosphatases regulate intracellular membrane traffic by binding specific effector proteins. The yeast Rab Sec4p plays multiple roles in the polarized transport of post-Golgi vesicles to, and their subsequent fusion with, the plasma membrane, suggesting the involvement of several effectors. Yet, only one Sec4p effector has been documented to date: the exocyst protein Sec15p. The exocyst is an octameric protein complex required for tethering secretory vesicles, which is a prerequisite for membrane fusion. In this study, we describe the identification of a second Sec4p effector, Sro7p, which is a member of the lethal giant larvae tumor suppressor family. Sec4-GTP binds to Sro7p in cell extracts as well as to purified Sro7p, and the two proteins can be coimmunoprecipitated. Furthermore, we demonstrate the formation of a ternary complex of Sec4-GTP, Sro7p, and the t-SNARE Sec9p. Genetic data support our conclusion that Sro7p functions downstream of Sec4p and further imply that Sro7p and the exocyst share partially overlapping functions, possibly in SNARE regulation.     

In Vitro Reconstitution of Rab GTPase-dependent Vesicle Clustering by the Yeast Lethal Giant Larvae/Tomosyn Homolog,Sro7

Intracellular traffic in yeast between the Golgi and the cell surface is mediated by vesicular carriers that tether and fuse in a fashion that depends on the function of the Rab GTPase, Sec4. Overexpression of either of two Sec4 effectors, Sro7 or Sec15, results in the formation of a cluster of post-Golgi vesicles within the cell. Here, we describe a novel assay that recapitulates post-Golgi vesicle clustering in vitro utilizing purified Sro7 and vesicles isolated from late secretory mutants. We show clustering in vitro closely replicates the in vivo clustering process as it is highly dependent on both Sro7 and GTP-Sec4. We also make use of this assay to characterize a novel mutant form of Sro7 that results in a protein that is specifically defective in vesicle clustering both in vivo and in vitro. We show that this mutation acts by effecting a conformational change in Sro7 from the closed to a more open structure. Our analysis demonstrates that the N-terminal propeller needs to be able to engage the C-terminal tail for vesicle clustering to occur. Consistent with this, we show that occupancy of the N terminus of Sro7 by the t-SNARE Sec9, which results in the open conformation of Sro7, also acts to inhibit vesicle cluster formation by Sro7. This suggests a model by which a conformational switch in Sro7 acts to coordinate Rab-mediated vesicle tethering with SNARE assembly by requiring a single conformational state for both of these processes to occur.     

The yeast tumor suppressor homologue Sro7p is required for targeting of the sodium pumping ATPase to the cell surface

The SRO7/SOP1 encoded tumor suppressor homologue of Saccharomyces cerevisiae is required for maintenance of ion homeostasis in cells exposed to NaCl stress. Here we show that the NaCl sensitivity of the sro7Delta mutant is due to defective sorting of Ena1p, the main sodium pump in yeast. On exposure of sro7Delta mutants to NaCl stress, Ena1p fails to be targeted to the cell surface, but is instead routed to the vacuole for degradation via the multivesicular endosome pathway. SRO7-deficient mutants accumulate post-Golgi vesicles at high salinity, in agreement with a previously described role for Sro7p in late exocytosis. However, Ena1p is not sorted into these post-Golgi vesicles, in contrast to what is observed for the vesicles that accumulate when exocytosis is blocked in sec6-4 mutants at high salinity. These observations imply that Sro7p has a previously unrecognized role for sorting of specific proteins into the exocytic pathway. Screening for multicopy suppressors identified RSN1, encoding a transmembrane protein of unknown function. Overexpression of RSN1 restores NaCl tolerance of sro7Delta mutants by retargeting Ena1p to the plasma membrane. We propose a model in which blocked exocytic sorting in sro7Delta mutants, gives rise to quality control-mediated routing of Ena1p to the vacuole.     

Novel proteins interacting with peroxisomal protein receptor PEX7 in Arabidopsis thaliana

Peroxisomal matrix protein transport relies on 2 cytosolic receptors, PEX5 and PEX7, which import peroxisomal targeting signal type 1 (PTS1) and PTS2-containing proteins, respectively. To better understand the transport mechanism of PEX7, we isolated PEX7 complexes using proteomics. We identified PEX5 as well as PTS1- and PTS2-containing proteins within the complex, thereby confirming the interaction between PEX5 and PEX7 during cargo transport that had been previously characterized by biochemical approaches. In addition, a chaperone T-complex and 2 small Rab GTPases were identified. We recently reported that the RabE1c is involved in the degradation of the PEX7 when abnormal PEX7 is accumulated on the peroxisomal membrane. This study expands our knowledge on the transport machinery via PEX7 by identifying both known and novel PEX7-interacting proteins and thus is helpful for further investigation of the regulation of the peroxisomal protein receptor during its translocation.     

Sorting of yeast membrane proteins into an endosome-to-Golgi pathway involves direct interaction of their cytosolic domains with Vps35p

Resident late-Golgi membrane proteins in Saccharomyces cerevisiae are selectively retrieved from a prevacuolar-endosomal compartment, a process dependent on aromatic amino acid-based sorting determinants on their cytosolic domains. The formation of retrograde vesicles from the prevacuolar compartment and the selective recruitment of vesicular cargo are thought to be mediated by a peripheral membrane retromer protein complex. We previously described mutations in one of the retromer subunit proteins, Vps35p, which caused cargo-specific defects in retrieval. By genetic and biochemical means we now show that Vps35p directly associates with the cytosolic domains of cargo proteins. Chemical cross-linking, followed by coimmunoprecipitation, demonstrated that Vps35p interacts with the cytosolic domain of A-ALP, a model late-Golgi membrane protein, in a retrieval signal-dependent manner. Furthermore, mutations in the cytosolic domains of A-ALP and another cargo protein, Vps10p, were identified that suppressed cargo-specific mutations in Vps35p but did not suppress the retrieval defects of a vps35 null mutation. Suppression was shown to be due to an improvement in protein sorting at the prevacuolar compartment. These data strongly support a model in which Vps35p acts as a "receptor" protein for recognition of the retrieval signal domains of cargo proteins during their recruitment into retrograde vesicles.     

Sro9, a Multicopy Suppressor of the Bud Growth Defect in the Saccharomyces Cerevisiae Rho3-Deficient Cells, Shows Strong Genetic Interactions with Tropomyosin Genes, Suggesting Its Role in Organization of the Actin Cytoskeleton

RHO3 encodes a Rho-type small GTPase in the yeast Saccharomyces cerevisiae and is involved in the proper organization of the actin cytoskeleton required for bud growth. SRO9 (YCL37c) was isolated as a multicopy suppressor of a rho3δ mutation. An Sro9p domain required for function is similar to a domain in the La protein (an RNA-binding protein). Disruption of SRO9 did not affect vegetative growth, even with the simultaneous disruption of an SRO9 homologue, SRO99. However, sro9δ was synthetically lethal with a disruption of TPM1, which encodes tropomyosin; sro9δ tpm1δ cells did not distribute cortical actin patches properly and lysed. We isolated TPM2, the other gene for tropomyosin, as a multicopy suppressor of a tpm1δ sro9δ double mutant. Genetic analysis suggests that TPM2 is functionally related to TPM1 and that tropomyosin is important but not essential for cell growth. Overexpression of SRO9 suppressed the growth defect in tpm1δ tpm2δ cells, disappearance of cables of actin filaments in both rho3δ cells and tpm1δ cells, and temperature sensitivity of actin mutant cells (act1-1 cells), suggesting that Sro9p has a function that overlaps or is related to tropomyosin function. Unlike tropomyosin, Sro9p does not colocalize with actin cables but is diffusely cytoplasmic. These results suggest that Sro9p is a new cytoplasmic factor involved in the organization of actin filaments.     

The Sec1/Munc18 Protein Groove Plays a Conserved Role in Interaction with Sec9p/SNAP‐25

The Sec1/Munc18 (SM) proteins constitute a conserved family with essential functions in SNARE‐mediated membrane fusion. Recently, a new protein–protein interaction site in Sec1p, designated the groove, was proposed. Here, we show that a sec1 groove mutant yeast strain, sec1(w24), displays temperature‐sensitive growth and secretion defects. The yeast Sec1p and mammalian Munc18‐1 grooves were shown to play an important role in the interaction with the SNAREs Sec9p and SNAP‐25b, respectively. Incubation of SNAP‐25b with the Munc18‐1 groove mutant resulted in a lag in the kinetics of SNARE complex assembly in vitro when compared with wild‐type Munc18‐1. The SNARE regulator SRO7 was identified as a multicopy suppressor of sec1(w24) groove mutant and an intact Sec1p groove was required for the plasma membrane targeting of Sro7p–SNARE complexes. Simultaneous inactivation of Sec1p groove and SRO7 resulted in reduced levels of exocytic SNARE complexes. Our results identify the groove as a conserved interaction surface in SM proteins. The results indicate that this structural element is important for interactions with Sec9p/SNAP‐25 and participates, in concert with Sro7p, in the initial steps of SNARE complex assembly.      

Lethal giant larvae proteins interact with the exocyst complex and are involved in polarized exocytosis

The tumor suppressor lethal giant larvae (Lgl) plays a critical role in epithelial cell polarization. However, the molecular mechanism by which Lgl carries out its functions is unclear. In this study, we report that the yeast Lgl proteins Sro7p and Sro77p directly interact with Exo84p, which is a component of the exocyst complex that is essential for targeting vesicles to specific sites of the plasma membrane for exocytosis, and that this interaction is important for post-Golgi secretion. Genetic analyses demonstrate a molecular pathway from Rab and Rho GTPases through the exocyst and Lgl to SNAREs, which mediate membrane fusion. We also found that overexpression of Lgl and t-SNARE proteins not only improves exocytosis but also rescues polarity defects in exocyst mutants. We propose that, although Lgl is broadly distributed in the cells, its localized interaction with the exocyst and kinetic activation are important for the establishment and reenforcement of cell polarity.     

Yeast homologues of lethal giant larvae and type V myosin cooperate in the regulation of Rab-dependent vesicle clustering and polarized exocytosis

Lgl family members play an important role in the regulation of cell polarity in eukaryotic cells. The yeast homologues Sro7 and Sro77 are thought to act downstream of the Rab GTPase Sec4 to promote soluble N-ethylmaleimide-sensitive factor adaptor protein receptor (SNARE) function in post-Golgi transport. In this article, we characterize the interaction between Sro7 and the type V myosin Myo2 and show that this interaction is important for two distinct aspects of Sro7 function. First, we show that this interaction plays a positive role in promoting the polarized localization of Sro7 to sites of active growth. Second, we find evidence that Myo2 negatively regulates Sro7 function in vesicle clustering. Mutants in either Myo2 or Sro7 that are defective for this interaction show hypersensitivity to Sro7 overexpression, which results in Sec4-dependent accumulation of large groups of vesicles in the cytoplasm. This suggests that Myo2 serves a dual function, to both recruit Sro7 to secretory vesicles and inhibit its Rab-dependent tethering activity until vesicles reach the plasma membrane. Thus Sro7 appears to coordinate the spatial and temporal nature of both Rab-dependent tethering and SNARE-dependent membrane fusion of exocytic vesicles with the plasma membrane.     

p24 proteins, intracellular trafficking, and behavior: Drosophila melanogaster provides insights and opportunities

The p24 transmembrane proteins, also known as EMP24/GP25 (endomembrane protein precursor of 24kD (Schimmoller et al., 1995)) proteins, are components of coat protein (COP)-coated vesicles and are present in species as diverse as fungi, plants, flies, worms, and mammals, indicating that they have important conserved functions. Genetic, molecular, and biochemical characterization of these proteins and the loci that encode them has provided insights into their potential cellular roles, including postulated functions in vesicle cargo protein selection and sorting, COPI and COPII vesicle formation and budding, and quality control of proteins that mature through the secretory pathway. Recently, the first mutations in a Drosophila melanogaster p24 gene have been isolated and characterized. These alleles produce an interesting behavioral phenotype in females, affecting their ability to oviposit. This identification and mutant characterization of a p24 locus in Drosophila will pave the way for a better understanding of cell-type-specific functions and interactions among p24 proteins.     

A Highlights from MBoC Selection: Structural basis for recognition of the Sec4 Rab GTPase by its effector,the Lgl/tomosyn homologue,Sro7

Members of the tomosyn/Lgl/Sro7 family play important roles in vesicle trafficking and cell polarity in eukaryotic cells. The yeast homologue, Sro7, is believed to act as a downstream effector of the Sec4 Rab GTPase to promote soluble N-ethylmaleimide–sensitive factor adaptor protein receptor (SNARE) assembly during Golgi-to–cell surface vesicle transport. Here we describe the identification of a Sec4 binding site on the surface of Sro7 that is contained within a cleft created by the junction of two adjacent β-propellers that form the core structure of Sro7. Computational docking experiments suggested four models for interaction of GTP-Sec4 with the Sro7 binding cleft. Further mutational and biochemical analyses confirmed that only one of the four docking arrangements is perfectly consistent with our genetic and biochemical interaction data. Close examination of this docking model suggests a structural basis for the high substrate and nucleotide selectivity in effector binding by Sro7. Finally, analysis of the surface variation within the homologous interaction site on tomosyn-1 and Lgl-1 structural models suggests a possible conserved Rab GTPase effector function in tomosyn vertebrate homologues.     

Proteome analysis of microtubule-associated proteins and their interacting partners from mammalian brain

The microtubule (MT) cytoskeleton is essential for a variety of cellular processes. MTs are finely regulated by distinct classes of MT-associated proteins (MAPs), which themselves bind to and are regulated by a large number of additional proteins. We have carried out proteome analyses of tubulin-rich and tubulin-depleted MAPs and their interacting partners isolated from bovine brain. In total, 573 proteins were identified giving us unprecedented access to brain-specific MT-associated proteins from mammalian brain. Most of the standard MAPs were identified and at least 500 proteins have been reported as being associated with MTs. We identified protein complexes with a large number of subunits such as brain-specific motor/adaptor/cargo complexes for kinesins, dynein, and dynactin, and proteins of an RNA-transporting granule. About 25% of the identified proteins were also found in the synaptic vesicle proteome. Analysis of the MS/MS data revealed many posttranslational modifications, amino acid changes, and alternative splice variants, particularly in tau, a key protein implicated in Alzheimer’s disease. Bioinformatic analysis of known protein–protein interactions of the identified proteins indicated that the number of MAPs and their associated proteins is larger than previously anticipated and that our database will be a useful resource to identify novel binding partners.     

A cell-specific transgenic approach in Xenopus reveals the importance of a functional p24 system for a secretory cell

The p24alpha, -beta, -gamma, and -delta proteins are major multimeric constituents of cycling endoplasmic reticulum-Golgi transport vesicles and are thought to be involved in protein transport through the early secretory pathway. In this study, we targeted transgene overexpression of p24delta2 specifically to the Xenopus intermediate pituitary melanotrope cell that is involved in background adaptation of the animal and produces high levels of its major secretory cargo proopiomelanocortin (POMC). The transgene product effectively displaced the endogenous p24 proteins, resulting in a melanotrope cell p24 system that consisted predominantly of the transgene p24delta2 protein. Despite the severely distorted p24 machinery, the subcellular structures as well as the level of POMC synthesis were normal in these cells. However, the number and pigment content of skin melanophores were reduced, impairing the ability of the transgenic animal to fully adapt to a black background. This physiological effect was likely caused by the affected profile of POMC-derived peptides observed in the transgenic melanotrope cells. Together, our results suggest that in the early secretory pathway an intact p24 system is essential for efficient secretory cargo transport or for supplying cargo carriers with the correct protein machinery to allow proper secretory protein processing.     

Molecular Dissection of Erv26p Identifies Separable Cargo Binding and Coat Protein Sorting Activities

Efficient export of secretory alkaline phosphatase (ALP) from the endoplasmic reticulum depends on the conserved transmembrane sorting adaptor Erv26p/Svp26p. In the present study we investigated the mechanism by which Erv26p couples pro-ALP to the coat protein complex II (COPII) export machinery. Site-specific mutations were introduced into Erv26p, and mutant proteins were assessed in cell-free assays that monitor interactions with pro-ALP cargo and packaging into COPII vesicles. Mutations in the second and third loop domains of Erv26p inhibited interaction with pro-ALP, whereas mutations in the C-terminal tail sequence influenced incorporation into COPII vesicles and subcellular distribution. Interestingly mutations in the second loop domain also influenced Erv26p homodimer associations. Finally we demonstrated that Ktr3p, a cis-Golgi-localized mannosyltransferase, also relies on Erv26p for efficient COPII-dependent export from the endoplasmic reticulum. These findings demonstrate that Erv26p acts as a protein sorting adaptor for a variety of Type II transmembrane cargo proteins and requires domain-specific interactions with both cargo and coat subunits to promote efficient secretory protein transport.Anterograde transport in the eukaryotic secretory pathway is initiated by the formation of COPII²-coated vesicles that emerge from transitional ER sites. The COPII coat, which consists of the small GTPase Sar1p, Sec23/24 complex, and Sec13/31 complex, selects vesicle cargo through recognition of export signals and forms ER-derived vesicles through assembly of an outer layer cage structure (1, 2). Cytoplasmically exposed ER export signals have been identified in secretory cargo including the C-terminal dihydrophic and diacidic motifs (3, 4). Structural studies indicate that the Sec24p subunit of the COPII coat contains distinct binding sites for some of the molecularly defined export signals (5, 6). Thus a cycle of cargo-coat interactions regulated by the Sar1p GTPase directs anterograde movement of secretory proteins into ER-derived transport vesicles (7).Although many secretory proteins contain known export signals that interact directly with COPII subunits, the diverse array of secretory cargo that depends on this export route requires additional machinery for efficient collection of all cargo into COPII vesicles (1). For instance certain soluble secretory proteins as well as transmembrane cargo require protein sorting adaptors for efficient ER export. These membrane-spanning adaptors, or sorting receptors, interact directly with secretory cargo and with coat subunits to efficiently couple cargo to the COPII budding machinery. For example, ERGIC-53 acts as a protein sorting adaptor for several glycoproteins and has a large N-terminal lumenal domain that interacts with secretory proteins including blood coagulation factors, cathepsins, and α₁-antitrypsin (8–10). The cytoplasmic C-terminal tail of ERGIC-53 contains a diphenylalanine export signal that is necessary for COPII export as well as a dilysine motif required for COPI-dependent retrieval to the ER (11). Additional ER vesicle proteins identified in yeast have been shown to interact with the COPII coat as well as specific secretory proteins (12). For example Erv29p acts as a protein sorting adaptor for the soluble secretory proteins glyco-pro-α-factor and carboxypeptidase Y (13). Erv29p also contains COPII and COPI sorting signals that shuttle the protein between ER and Golgi compartments. More recently Erv26p was identified as a cargo receptor that escorts the pro-form of secretory alkaline phosphatase (ALP) into COPII-coated vesicles (14).Although COPII sorting receptors have been identified, the molecular mechanisms by which these receptors link cargo to coat remain poorly understood. Moreover it is not clear how cargo binding is regulated to promote interaction in the ER and then trigger dissociation in the Golgi complex. We have shown previously that Erv26p binds to pro-ALP and is required for efficient export of this secretory protein from the ER (14). Therefore specific lumenal regions of Erv26p are proposed to interact with pro-ALP, whereas cytosolically exposed sorting signals are presumably recognized and bound by coat subunits. To gain insight on the molecular contacts required for Erv26p sorting function, we undertook a systematic mutational analysis of this multispanning membrane protein. After generating a series of Erv26p mutants, we observed that mutation of specific residues in the third loop domain affect pro-ALP interaction and that residues in the C-terminal cytosolic tail are required for COPII and COPI transport. Finally mutation of residues in the second loop domain influenced Erv26p homodimer formation and sorting activity.     

Proteomics of endoplasmic reticulum-Golgi intermediate compartment (ERGIC) membranes from brefeldin A-treated HepG2 cells identifies ERGIC-32, a new cycling protein that interacts with human Erv46

Cycling proteins play important roles in the organization and function of the early secretory pathway by participating in membrane traffic and selective transport of cargo between the endoplasmic reticulum (ER), the intermediate compartment (ERGIC), and the Golgi. To identify new cycling proteins, we have developed a novel procedure for the purification of ERGIC membranes from HepG2 cells treated with brefeldin A, a drug known to accumulate cycling proteins in the ERGIC. Membranes enriched 110-fold over the homogenate for ERGIC-53 were obtained and analyzed by mass spectrometry. Major proteins corresponded to established and putative cargo receptors and components mediating protein maturation and membrane traffic. Among the uncharacterized proteins, a 32-kDa protein termed ERGIC-32 is a novel cycling membrane protein with sequence homology to Erv41p and Erv46p, two proteins enriched in COPII vesicles of yeast. ERGIC-32 localizes to the ERGIC and partially colocalizes with the human homologs of Erv41p and Erv46p, which mainly localize to the cis-Golgi. ERGIC-32 interacts with human Erv46 (hErv46) as revealed by covalent cross-linking and mistargeting experiments, and silencing of ERGIC-32 by small interfering RNAs increases the turnover of hErv46. We propose that ERGIC-32 functions as a modulator of the hErv41-hErv46 complex by stabilizing hErv46. Our novel approach for the isolation of the ERGIC from BFA-treated cells may ultimately lead to the identification of all proteins rapidly cycling early in the secretory pathway.