Syntaxin 5: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References
Gene name - Syntaxin 5
Synonyms - Sed5
Cytological map position - 35F8
Function - Golgi traffic and assembly
Keywords - Golgi vesicle transport and fusion, spermatogenesis, cell division
Symbol - Syx5
FlyBase ID: FBgn0011708
Genetic map position - 2-
Classification - t-SNARE coiled-coil homology domain
Cellular location - transmembrane in the Golgi apparatus
|Recent literature||Satoh, T., Nakamura, Y. and Satoh, A. K. (2016). The roles of Syx5 in Golgi morphology and rhodopsin transport in Drosophila photoreceptors. Biol Open [Epub ahead of print]. PubMed ID: 27591190
SNAREs (SNAP receptors) are the key components of protein complexes that drive membrane fusion. This study reports the function of a SNARE, Syntaxin 5 (Syx5), in the development of photoreceptors in Drosophila In wild type photoreceptors, Syx5 localizes to cis-Golgi, along with cis-Golgi markers, Rab1, and GM130. It was observed that Syx5-deficient photoreceptors shows notable accumulation of these cis-Golgi markers accompanying drastic accumulation of vesicles between ER and Golgi cisternae. Extensive analysis of Rh1 (rhodopsin 1) trafficking revealed that in Syx5-deficient photoreceptors, Rh1 is exported from the ER with normal kinetics, retained in cis-Golgi region along with GM130 for a prolonged period, then subsequently degraded presumable by endoplasmic-reticulum-associated protein degradation (ERAD) after retrieved to the ER. Unlike a previous report of Rab6-deficient photoreceptors-where two apical transport pathways are specifically inhibited-vesicle transport pathways to all of plasma membrane domains are inhibited in Syx5-deficient photoreceptors, implying that Rab6 and Syx5 are acting in different steps of intra-Golgi transport. These results indicate that Syx5 is crucial for membrane protein transport, presumably during ER-derived vesicle fusion to form cis-Golgi cisternae.
Syntaxin 5 is a Golgi-localized SNARE protein that has been shown to be required for ER-Golgi traffic in yeast (Dascher, 1994) and Golgi reassembly following cell division in mammalian cells (Rabouille, 1998). The Drosophila ortholog, Syx5, like its mammalian and yeast counterparts is localized to the Drosophila Golgi and binds to alpha-SNAP. Null mutations in Syx5 are larval lethal and demonstrate impaired transport of vesicles through the secretory pathway. A hypomorphic allele of Syx5 results in impenetrant lethality, and escaping adult flies are male sterile. The male sterility results both from failure of germ cells to complete cytokinesis and from defects in spermatid elongation and maturation. Together, these results show that Syx5 is required for the proper function of the Golgi apparatus and that an efficiently functioning Golgi apparatus is required for the steps leading to the completion of cytokinesis and formation of mature sperm (H. Xu, 2002).
Transport of membranes and membrane proteins within the cell requires the interaction of SNARE proteins on the transport vesicle with their cognate SNARE partners on the target membrane, and this binding appears necessary to achieve membrane fusion. The SNARE proteins, so named because they form a stable complex that acts as the receptor for the soluble NSF attachment protein alpha-SNAP (hence SNAP Receptors), are composed of three protein families: VAMP, syntaxin, and SNAP-25. The formation of this strong coiled-coil complex is thought to draw the vesicle and target membranes into apposition, and in so doing may provide the energy needed to cause membrane fusion (H. Xu, 2002 and references therein).
Although the role of SNARE proteins in secretion is generally accepted, a large number of different SNARE protein isoforms have been identified that appear to have specific subcellular distributions. This has led to the idea that membrane fusion at each step in the secretory pathway may be mediated by the interactions of a unique set of SNAREs. A recent comparison of the partially completed human and Drosophila genomes with those of Caenorhabditis elegans and Saccharomyces cerevisiae has revealed that, while yeast, worms, and flies have roughly the same number of SNAREs, humans have significantly more SNARE proteins (Bock, 2001). This suggests that mammals may have evolved unique forms of the SNAREs for specialized purposes. However, certain ancestral forms of the SNAREs, such as syntaxin 5 (also known as Sed5p), have orthologs that are present in all organisms (H. Xu, 2002).
Sed5 was first identified by Hardwick and Pelham (1992) as a multicopy suppressor of the lethal phenotype that arose from the lack of the yeast HDEL receptor ERD2. Their studies revealed that Sed5p participates in vesicular traffic between the ER and Golgi. The mammalian ortholog, called syntaxin 5, was found to have a cis-Golgi distribution (Bennett, 1993) consistent with a role in this compartment. Similarly, the Drosophila ortholog, when expressed in mammalian cells, also localized to a perinuclear compartment (Banfield, 1994). Subsequent studies revealed that overexpression of a truncated form of mammalian syntaxin 5 lacking the transmembrane domain (Dascher, 1994) or microinjection of syntaxin 5-specific antibodies (Rowe, 1998) blocked the transport of vesicular stomatitis virus glycoprotein in a pre-Golgi intermediate compartment. Together, these results suggest that syntaxin 5 is required for the fusion of the carrier vesicles at the cis-face of the Golgi complex. In addition, syntaxin 5 has been implicated (Rabouille, 1998) in the reassembly of the Golgi apparatus from mitotic fragments following cell division (H. Xu, 2002).
In single cell systems, there is clear evidence that Sed5/syntaxin 5 (Syx5) functions in the Golgi complex, but the overall function of Syx5 in the development of a multicellular organism is not known. The Drosophila Syx5 locus has been characterized, and, like its mammalian and yeast counterparts, Syx5 is localized to the Golgi complex. As in mammals, Syx5 binds to alpha-SNAP in both two-hybrid and in vitro assays. As well, Syx5 interacts genetically with N-ethylmaleimide sensitive factor (NSF). A null mutation within the Syx5 gene has been characterized; the absence of Syx5 protein causes lethality early during the first larval instar. Moreover, using the polarized epithelial cells in the embryonic salivary gland as a model, it has been found that null mutations display defects in apical transport. Interestingly, hypomorphic combinations of Syx5 mutations lead to male sterility, and this is due to a failure in both cytokinesis and sperm maturation. Together, these results show that Syx5 is required for normal membrane protein transport and development. Moreover, these results suggest that cytokinesis is dependent on a functional Golgi complex and that this process is particularly sensitive to levels of Syx5 (H. Xu, 2002).
To search for novel SNARE proteins in Drosophila, advantage was taken of the fact that members of the syntaxin family of SNARE proteins are able to bind directly to alpha-SNAP. Therefore a two-hybrid screen of an ovarian cDNA library was performed using Drosophila alpha-SNAP as bait. In this screen, five independent, strongly positive clones were identified that were identical to the Drosophila homolog of syntaxin 5, Syx5. All five cDNAs isolated were partial and all contained sequences corresponding to the helical region of the protein (termed H3 in syntaxin family members) closest to the transmembrane domain. The H3 helical domain is involved in the formation of coiled-coil interactions with partner SNAREs and in mammalian syntaxin-1 is the region to which alpha-SNAP binds (H. Xu, 2002).
Null mutations in Syx5 cause an accumulation of membrane proteins in intracellular compartments and are larval lethal, providing evidence for a required role for membrane traffic during early stages of development. This suggests that Syx5 is important for transport of membrane proteins and, further, that blockade of the ER-Golgi traffic by Syx5 deficiency likely results in the accumulation of secretory proteins in ER-derived transport vesicles. Moreover, the early larval lethality probably arises due to depletion of the maternally supplied Syx5, leading to the arrest of a variety of signaling pathways and physiological controls that require membrane protein synthesis and transport. Unexpectedly, however, these studies have also revealed a role for Syx5 function in animal cell cytokinesis and spermatid differentiation (H. Xu, 2002).
The process of spermatogenesis in Drosophila has been the subject of extensive analysis and mutations have been identified that affect many stages in sperm development. During spermatogenesis in Drosophila, germ cells undergo four mitotic divisions and two meiotic divisions, each with incomplete cytokinesis. These divisions led to the formation of syncytial cysts of 64 spermatids connected to each other by 63 ring canals. Each testis contains a number of cysts at different stages in maturation (H. Xu, 2002 and references therein).
Within each syncytium, the spermatids form bundles that elongate the length of the testis, but then must acquire their own cell membranes in a process called individualization. Individualization occurs when a fiber-rich structure called the investment cone surrounds each elongated spermatid and progresses toward the caudal end of the spermatids, excluding all of the organelles into a so-called waste bag and encasing each spermatid in its own membrane (H. Xu, 2002).
By analyzing the phenotype of hypomorphic alleles of Syx5, it has been found that the developmental steps most sensitive to the proper function of Syx5 are the processes of cytokinesis and sperm maturation within the male germline. Syx5, an EP line [EP(2)2313] was obtained that resulted from an insertion into the Syx5 promoter region. The combination of the EP allele with a amber mutant dSyx5AR113 functions as a hypomorph, which allows for adult escapers. The EP(2)2313/dSyx5AR113 hypomorph together with flies expressing increasing levels of Syx5 from the EP chromosome (driven by da-GAL4 and act-GAL4) represent, in effect, an allelic series that demonstrates a requirement for Syx5 at multiple stages of spermatogenesis. EP(2)2313/dSyx5AR113 flies are defective in cytokinesis, the formation of elongated spermatid bundles, and sperm individualization. Expression of Syx5 under da-GAL4 control rescues both cytokinesis and bundle elongation, yet mature individualized sperm fail to form. Expression of Syx5 at the highest level (from act-GAL4) results in the production of motile sperm. These results imply that different levels of Syx5-dependent secretion are required for at least three distinct processes in this tissue: meiotic cytokinesis, outgrowth of membranes to form elongated spermatid cysts, and individualization of sperm. Interestingly, in situ hybridization studies revealed that Syx5 expression is highest in primary spermatocytes, suggesting that the proteins are produced at sufficient levels to persist throughout individualization (H. Xu, 2002).
Spermatid bundle elongation and individualization represent two aspects of male germ cell development that are accompanied by extensive plasma membrane remodeling. During the elongation of spermatids, a specialized part of the germ cell cytoplasm called the fusome passes through the intercellular bridges, or ring canals, which are composed of the actin-binding proteins anillin and the septins. The fusome consists of highly branched membranous structures, probably contiguous with ER, and is marked by the presence of membrane skeletal proteins alpha-spectrin and adducin. It is possible that the fusome may provide membrane for bundle elongation. Alternatively, a source for this membrane may be the many Lava lamp-positive vesicles present along the elongating spermatid tails that are disrupted in Syx5 mutants. Since septins play roles in secretion, perhaps they coordinate fusion of these Golgi-derived vesicles at the site of membrane growth during bundle formation (H. Xu, 2002 and references therein).
Separation of syncytial spermatids into individual sperm is achieved by the action of an individualization complex (IC) composed of F-actin-rich investment cones that form around the nuclei of elongated spermatids and traverse the length of the spermatid bundles. This process invests each cell with its own plasma membrane and simultaneously strips away excess cytoplasmic material not needed by mature sperm. The discarded material is deposited in so-called waste bags that contain, among other components, investment cones, ring canals, and fusome material. Proper IC function requires a large number of genes, including jaguar, which encodes an unconventional myosin. ICs formed and progressed at least part way down the bundles of da-GAL4;EP(2)2313/dSyx5AR113 spermatids. However, few waste bags were observed and no mature sperm formed. Flies expressing higher levels of Syx5 (from the act-GAL4 driver) have motile sperm and are fertile. Syx5 is thus the first secretory protein shown to play a role in the elaborate membrane remodeling events required to produce individual sperm (H. Xu, 2002).
While a requirement for membrane addition during animal cell cytokinesis is only an emerging concept, it has long been known that cytokinesis in plants requires membrane traffic for the formation of a membrane plate, called the phragmoplast, to separate the daughter cells (for a review, see Verma, 2001). Unlike animal cells in which fission is mediated in part by an actomyosin-based constrictive ring, the rigid cell wall precludes the action of constriction to divide the cytoplasm. In Arabidopsis, two proteins called KNOLLE and KEULE interact to promote vesicle fusion during cytokinesis. KNOLLE is a cytokinesis-specific syntaxin homolog, and KEULE is related to the syntaxin-binding protein Sec1, suggesting that KNOLLE proteins may serve as the target SNARE proteins during membrane addition. Interestingly, centrifugal growth of the phragmoplast plate is thought to occur by the addition of membranes derived from the Golgi complex, since the fungal metabolite brefeldin A inhibits it, apparently by eliminating the supply of vesicles from the Golgi complex (H. Xu, 2002 and reference therein).
To date, the best evidence of a role for membrane addition during the division of animal cells has been the demonstration that plasma membrane syntaxin proteins in sea urchin and C. elegans are required for this process. In sea urchins, introduction of Botulinum C1 neurotoxin, a protease that cleaves certain mammalian syntaxins, blocked cytokinesis, although the specific target for this toxin was not identified (Conner, 1999). In C. elegans, double-strand RNA inhibition (dsRNAi) was used to test the function of syntaxin isoforms, and it was found that inhibition of syntaxin 4, the worm ortholog of mammalian syntaxin 1, led to multinucleated cells (Jantsch-Plunger, 1999). Interestingly, of the eight syntaxins present in C. elegans, dsRNAi injections of only two leads to embryonic lethality -- syntaxin 4 and syntaxin 3 (the C. elegans ortholog of Syx5). However, the latter had a complex and pleiotropic phenotype and it was not analyzed for its effect on cytokinesis (H. Xu, 2002).
Further supporting the requirement of efficient Golgi function for spermatocyte mitosis and maturation comes from a screen for mutations in spermatogenesis. Null mutations in four way stop (fws), the Drosophila ortholog of cog5, give a virtually identical phenotype to the hypomorphic mutation in syntaxin 5. Cog5 appears to act as a molecular tether to enhance Golgi transport in vitro. The Drosophila Cog5 homolog is required for cytokinesis, polarized cell growth, and assembly of specialized Golgi architecture during spermatogenesis. Loss-of-function mutations in fws causes failure of cleavage furrow ingression in dividing spermatocytes and failure of cell elongation in differentiating spermatids and disrupts the formation and/or stability of the Golgi-based spermatid acroblast. Consistent with the lack of a growth defect in yeast lacking Cog5, adult flies lacking fws function were viable, although males were sterile. Fws protein localizes to Golgi structures throughout spermatogenesis. It is proposed that Fws may directly or indirectly facilitate efficient vesicle traffic through the Golgi to support rapid and extensive increases in cell surface area during spermatocyte cytokinesis and polarized elongation of differentiating spermatids (Farkas, 2003).
Taken together, these results suggest that SNARE-mediated membrane addition is required for cytokinesis in animal cells and support the notion that syntaxin proteins play an important role as target SNAREs for this new membrane, and specifically that a well characterized Golgi protein, Syntaxin 5 is involved in this process (H. Xu, 2002).
The source of the membranes involved in cytokinesis, however, has been less clear. As indicated above, phragmoplast formation occurs through the fusion of vesicles derived from the Golgi apparatus. Similarly, cellularization in Drosophila is also achieved through vesicles from the Golgi (Sisson, 2000). Recent studies in C. elegans have shown that incubation of embryos in brefeldin A led to an inhibition of the completion of cytokinesis. In this case, the cleavage furrow ingressed properly, but stalled and finally regressed (Skop, 2001). Interestingly, using FM1-43 labeling methods, the authors were able to demonstrate the accumulation of vesicles near the cleavage furrow in normal embryos, but showed an absence of such vesicles following brefeldin A treatment, suggesting that they may be derived from the Golgi. Finally, the recent discovery that a kinesin-like protein (Rab6-KIFL) that binds Rab6, a Golgi-localized Rab protein necessary for intra-Golgi transport (Hill, 2000), is localized to the narrow bridge linking dividing HeLa cells during late telophase, has implicated Golgi membranes in mammalian cell division (H. Xu, 2002).
Why would the Golgi complex represent an ideal source of membranes to carry out the task of building a membrane plate between dividing cells? (1) Golgi-derived vesicles can fuse with other Golgi complex compartments, a type of homotypic fusion necessary to generate a membrane de-novo. Indeed, during mitosis the Golgi complex is dissociated into mitotic vesicles that partition between the daughter cells and subsequently reassemble into new Golgi complexes. Interestingly, Syx5 is important in this reassembly, as antibodies against syntaxin 5 block reassembly in vitro (Rabouille, 1998). (2) Since the Golgi membranes must be partitioned between the two cells, their movement must be linked to the cell cycle. In fact, Rab6- KIFL may be responsible for partitioning the Golgi into the daughter cells and to the midbody between the daughter cells in telophase. (3) The Golgi complex is itself a plate-like structure and the formation of a sheet of membranes may be ideally achieved with vesicles of the lipid composition that makes up the flat stacks of the Golgi complex. The homotypic fusion of Golgi-derived vesicles at the narrow, constricted midbody region could contribute to furrow ingression, or form a sheetlike stucture akin to the phragmoplast that would sever the two cells and resolve cytokinesis. Future studies will be aimed at determining how cytokinesis is regulated and which other membrane trafficking proteins are involved (H. Xu, 2002).
One of the most significant morphogenic events in the development of Drosophila is the elongation of imaginal discs during puparium formation. This macroscopic event is accompanied by the formation of Golgi stacks from small Golgi larval clusters of vesicles and tubules that are present prior to the onset of disc elongation. The fly steroid hormone 20-hydroxyecdysone triggers both the elongation itself and the formation of Golgi stacks. Using mRNA in situ hybridisation, it has been shown that ecdysone triggers the upregulation of a subset of genes encoding Golgi-related proteins (such as dnsf1, dsec23, Syx5, and drab1) and downregulates the expression of others (such as dergic53, dbeta'COP, and drab6). The transcription factor Broad-complex, itself an "early" ecdysone target, mediates this regulation. The ecdysone-independent upregulation of dnsf1 and dsnap prior to the ecdysone peak leads to a precocious formation of large Golgi stacks. The ecdysone-triggered biogenesis of Golgi stacks at the onset of imaginal disc elongation offers the exciting possibility of advancing understanding of the relationship between gene expression and organelle biogenesis (Dunne, 2002).
To confirm the significance of the two-hybrid analysis that detected an interaction between alpha-SNAP and Syt5, a GST fusion protein was generated with a Syx5 cDNA lacking the transmembrane domain (Syx5delta) and the interaction of Syx5delta with increasing amounts of recombinant His6-alpha-SNAP was examined. Western blots of eluted protein were probed with anti-GST to confirm the presence of GST-Syx5delta on the glutathione-agarose beads, and with anti-alpha-SNAP to measure alpha-SNAP binding. Under nonsaturating binding conditions, alpha-SNAP binds in a dose-dependent manner. alpha-SNAP from adult fly homogenates is also able to bind to immobilized recombinant Syx5delta. These results establish that Syx5 acts as an alpha-SNAP receptor and define the H3 domain as the likely region for this interaction (H. Xu, 2002).
Another predicted function of a SNARE protein is its ability to interact with other members of the SNARE pathway. The phenotype that results from the overexpression of a dominant-negative form of Drosophila NSF2 at the developing wing margin has been described (Stewart, 2001). To determine whether Syx5 interacts genetically with NSF2, a single copy of a null mutation in Syx5 was introduced into the flies expressing dominant-negative NSF2 along the wing margin; a significant enhancement of wing notching was seen. This enhancement was as strong as any of the other known interactors identified previously (Stewart, 2001) and provides further evidence that Syx5 functions as a SNARE in vivo (H. Xu, 2002).
In situ hybridization was used to analyze the expression pattern of Syx5; the gene is broadly expressed throughout the developing embryo in virtually all tissues and is present at high levels in 1-h-old embryos, indicative of a significant maternal contribution (H. Xu, 2002).
In mammals, syntaxin 5 has been localized to a perinuclear compartment likely to be the cis portion of the Golgi complex (Bennett, 1993). To determine whether Syx5 in Drosophila behaves the same as its mammalian ortholog, it was determined whether antibodies against rat syntaxin 5 would recognize Drosophila Syx5. This antibody indeed recognizes an IPTG-inducible band that represents the GST-Syx5 fusion. This antibody was then used for immunocytochemistry on Schneider S2 cells. Anti-syntaxin 5 antibodies revealed a punctate, perinuclear pattern of staining that overlapped extensively with the Golgi marker p120. To further confirm this colocalization, the full-length cDNA was subcloned into the pRmHa-3 expression vector in frame with an N-terminal Myc epitope. This was transfected into S2 cells, and the cells were costained with antibodies against Myc and p120. Again, significant overlap of the Myc and p120 signals was seen. Hence, the Drosophila Syx5 protein, like its mammalian ortholog, predominantly resides in the Golgi complex (H. Xu, 2002).
The Syx5 gene maps near cornichon and extensive EMS mutagenic analysis of the locus has identified several lethal complementation groups that appear to be in the vicinity of the Syx5 gene. It was determined that complementation group l(2)35Ff was likely to correspond to Syt5. To determine whether any of the members of this complementation group contain mutations in Syx5, RT-PCR was used to amplify the Syx5 cDNAs from this group with four alleles (dSyx5AR113, dSyx5AE48, dSyx5AA73, and dSyx5AE73). Sequence analysis reveals that the line dSyx5AR113 contains an amber mutation at glutamine residue 153. This mutation would create a truncated N-terminal peptide that should not interact with the SNARE proteins or alpha-SNAP and would therefore be expected to be functionally null. Analysis of the homozygous or transheterozygous combinations of the complementation group revealed lethality during the first larval instar. This indicates that, although the molecular basis for the other mutations is not known, all are likely to be null or severe hypomorphic alleles (H. Xu, 2002).
Since Syx5 is implicated in the traffic of membranes and membrane proteins through the Golgi complex, exocytosis of an exogenous fluorescent transgene was monitored in Oregon R (Ore-R) and Syx5 mutant flies. For this purpose, UAS-mCD8-GFP flies were used. mCD8-GFP is a protein fusion between the murine lymphocyte receptor CD8 and EGFP. It accumulates at the plasma membrane of most cells, and in epithelial cells is found to accumulate at the apical membranes. To express the mCD8-GFP construct broadly, the UAS-mCD8-GFP and a GAL4 gene driven by the daughterless promoter (da-GAL4) were introduced into the dSyx5AR113 or wild type backgrounds. In the embryonic salivary gland of heterozygous dSyx5AR113/CyO flies, mCD8-GFP can be seen to accumulate on the apical surface during embryogenesis. In contrast, flies homozygous for the Syx5 mutation have little if any mCD8-GFP on the salivary gland apical surface and instead appear to accumulate fluorescence throughout the cytoplasm. Hence, Syx5 function appears necessary for proper transport through the secretory pathway (H. Xu, 2002).
In addition to the EMS alleles of Syx5, an EP line [EP(2)2313] was obtained that resulted from an insertion into the Syx5 promoter region. EP elements are modified P transposable elements that contain a GAL4 binding site and a weak promoter, allowing the directional expression of adjacent genes. EP(2)2313 is 803 bp upstream of the Syx5 ATG and only 46 bp upstream of the longest cDNA in the dbEST database. In addition, EP(2)2313 is in the correct orientation to permit overexpression by GAL4. The location of EP(2)2313 also suggested that it might be hypomorphic for Syx5. EP(2)2313 is homozygous lethal, but this lethality is likely due to second-site mutations or the effect of the EP on adjacent genes, since it is only semilethal in combination with the Syx5 mutant allele dSyx5AR113. When da-GAL4 is introduced into the EP(2)2313/Syx5AR113 background, expression of Syx5 is increased and the semilethality is fully rescued. Similar results were obtained by using da-GAL4 to rescue another allele of Syx5, Syx5AE48 as a transheterozygote with EP(2)2313. In contrast, da-GAL4 cannot rescue the lethality of the EP(2)2313 homozygotes, indicating that their lethality is not due to lack of Syx5. Hence, it appears that EP(2)2313 is hypomorphic for Syx5 (H. Xu, 2002).
The incomplete penetrance of the lethal phenotype exhibited by EP(2)2313/dSyx5AR113 permitted analysis of the adult escapers. Whereas the females appeared normal, the males were sterile when crossed to female escapers or Ore-R females, suggesting that Syx5 mutant flies have a defect in some aspect of spermatogenesis (H. Xu, 2002).
Initial examination of the testes of the Syx5 mutant flies revealed that they have no motile sperm and appear to be defective in spermatid elongation. In contrast to wild type testes, Syx5 mutant testes contained predominantly large, oval cysts and few elongated bundles. In situ hybridization was used to examine when during spermatogenesis Syx5 is expressed. Syx5 is expressed in the primary spermatocytes, but expression does not persist past the meiosis stage and is essentially absent from the elongating bundles (H. Xu, 2002).
Examination of the Golgi marker Lava lamp (Lva) reveals that, in those cysts that do undergo elongation, Lva staining appeared diffuse with a mixture of large and small punctate structures. In contrast, wild type flies display exclusively large punctate structures. These results indicate that spermatogenesis may be a developmental process that is particularly sensitive to the function of Syt5 (H. Xu, 2002).
Closer analysis of the spermatids reveals that more than 80% of those from the Syx5 mutant flies have abnormally large mitochondrial derivatives that are associated with multiple nuclei. Since mitochondria fuse together following meiosis, the presence of a large mitochondrial derivative surrounded by multiple nuclei is consistent with cells having failed to complete meiotic cytokinesis (H. Xu, 2002).
It is important to note that membrane fusion often occurs during the preparation of these cells for microscopy, causing nuclei to become colocalized. However, the presence of a single large mitochondrial derivative surrounded by nuclei is diagnostic of failed cytokinesis events, whereas multiple mitochondria and nuclei of the same size within a cell have arisen as an artifact in the unfixed squashed preparations. Typical mutant spermatids have an enlarged mitochondrial derivative associated with two or more nuclei (H. Xu, 2002).
Quantification of the cytokinesis failure obtained from examining a large number of cysts reveals that, of a total of 1361 mutant nuclei counted, the vast majority are present in multinucleated cells. In contrast, most cysts from control flies have mitochondrial derivatives and nuclei of approximately the same size and fewer than 0.5% of cysts contain multiple nuclei. Spermatids with 2 nuclei likely failed a single meiotic division while those with four probably failed to divide during both meioses. Occasionally, some spermatids are observed that have 8 nuclei, suggestive of an additional failure at an earlier mitotic division step. In addition, some spermatids have numbers of nuclei that could have arisen if a multinucleated parental cell divided unequally (H. Xu, 2002).
These phenotypes are similar to those observed in spermatids from several male meiosis mutants including four wheel drive (fwd) (Brill, 2000). fwd encodes a phosphatidylinositol 4-kinase that is required for completion of cytokinesis, particularly during the meiotic divisions, and the similarity of the two phenotypes suggests that Syx5 is also required for this process (H. Xu, 2002).
If the defects in cytokinesis are due to the lack of Syx5, then it should be possible to rescue this by increasing Syx5 expression. da-GAL4 was introduced into the EP(2)2313/dSyx5AR113 background to drive Syx5 expression from EP(2)2313 chromosome. It was first determined whether the EP line could lead to elevated Syx5 expression in the presence of the GAL4 driver. To measure this, a pair of PCR primers was generated, one from the EP element and one from the 3' end of the Syx5 gene, to measure transcripts generated from the EP. The EP primer would be included in transcripts that arose from the EP element when activated by GAL4 and would measure only those transcripts. As a control, primers from Syx5 from the 5' and 3' ends of the Syx5 coding sequence were generated. These would detect all Syx5 transcripts, since the primer sites are within both the wild type and EP-derived mRNAs. Indeed, using RT-PCR, fusion transcripts could be detected from the EP only when the da-GAL4 driver was present, but not in the control flies. Cysts from the da-GAL4;EP(2)2313/dSyx5AR113 flies that overexpress Syx5 reveal significant rescue of the multinucleated phenotype. Occasional cells with two nuclei are observed, but quantification of these data clearly reveals a nearly complete rescue of cytokinesis by Syx5 expression. Only 3.9% of the spermatids counted contained multiple nuclei (compared with 80% without rescue), and viability was completely restored (H. Xu, 2002).
In addition to a failure in cytokinesis, EP(2)2313/ dSyx5AR113 testes also exhibit an accumulation of unusual ovoid spermatid cysts. Expression of Syx5 from the EP chromosome with da-GAL4 rescues both the cytokinesis and these bundle elongation defects. Interestingly, few motile sperm were observed and the flies remained infertile. This suggests that either insufficient levels of Syx5 were reached to achieve complete rescue, or other unrelated causes were responsible for the late stage maturation defects. In support of the former idea, introducing the stronger actin-GAL4 driver into the EP(3)2313/dSyx5AR113 background achieves complete rescue of the spermatid maturation defect, resulting in fertile flies with motile sperm. Thus, strong expression of Syx5 from the EP is sufficient to rescue all spermatogenesis defects associated with mutations in dSyx5 (H. Xu, 2002).
The ERD2 gene, which encodes the yeast HDEL (His-Asp-Glu-Leu) receptor, is essential for growth. SED5, when present in multiple copies, enables cells to grow in the absence of Erd2p. Sequence analysis of SED5 reveals no significant homology with ERD2 or other known genes. Antibodies were raised to Sed5p which specifically recognize a 39-kD integral membrane protein. A stretch of hydrophobic residues at the COOH terminus is predicted to hold Sed5p on the cytoplasmic face of intracellular membranes. Cells that are depleted of Sed5p are unable to transport carboxypeptidase Y to the Golgi complex, and stop growing after a dramatic accumulation of ER membranes and vesicles. It is concluded that the SED5 gene is essential for growth and that Sed5p is required for ER to Golgi transport. When Sed5p is overexpressed the efficiency of ER to Golgi transport is reduced, vesicles accumulate, and cellular morphology is perturbed. Immunofluorescence studies reveal that the bulk of Sed5p is not found on ER membranes but on punctate structures throughout the cytoplasm, the number of which increases upon SED5 overexpression. It is suggested that Sed5p has an essential role in vesicular transport between ER and Golgi compartments and that it may itself cycle between these organelles (Hardwick, 1992).
The generation of transport vesicles at the endoplasmic reticulum (ER) depends on cytosolic proteins, which, in the form of subcomplexes (Sec23p/Sec24p; Sec13p/Sec31p) are recruited to the ER membrane by GTP-bound Sar1p and form the coat protein complex II (COPII). Using affinity chromatography and two-hybrid analyses, it has been found that the essential COPII component Sec24p, but not Sec23p, binds to the cis-Golgi syntaxin Sed5p. Sec24p/Sed5p interaction in vitro was not dependent on the presence of [Sar1p.GTP]. The binding of Sec24p to Sed5p is specific; none of the other seven yeast syntaxins bound to this COPII component. Whereas the interaction site of Sec23p is within the N-terminal half of the 926-aa-long Sec24p (amino acid residues 56-549), Sed5p binds to the N- and C-terminal halves of the protein. Destruction by mutagenesis of a potential zinc finger within the N-terminal half of Sec24p leads to a nonfunctional protein that is still able to bind Sec23p and Sed5p. Sec24p/Sed5p binding might be relevant for cargo selection during transport-vesicle formation and/or for vesicle targeting to the cis-Golgi (Peng, 1999).
To fuse transport vesicles with target membranes, proteins of the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) complex must be located on both the vesicle (v-SNARE) and the target membrane (t-SNARE). In yeast, four integral membrane proteins, Sed5, Bos1, Sec22 and Bet1, each probably contribute a single helix to form the SNARE complex that is needed for transport from endoplasmic reticulum to Golgi. This generates a four-helix bundle, which ultimately mediates the actual fusion event. How the anchoring arrangement of the four helices affects their ability to mediate fusion is examined in this study. Two populations of phospholipid bilayer vesicles were reconstituted, with the individual SNARE proteins distributed in all possible combinations between them. Of the eight non-redundant permutations of four subunits distributed over two vesicle populations, only one results in membrane fusion. Fusion only occurs when the v-SNARE Bet1 is on one membrane and the syntaxin heavy chain Sed5 and its two light chains, Bos1 and Sec22, are on the other membrane where they form a functional t-SNARE. Thus, each SNARE protein is topologically restricted by design to function either as a v-SNARE or as part of a t-SNARE complex (Parlati, 2000).
Sed5p is the only syntaxin family member required for protein transport through the yeast Golgi and it is known to bind up to nine other SNARE proteins in vivo. In vitro binding experiments arte described in which ternary and quaternary Sed5p-containing SNARE complexes were identified. The formation of SNARE complexes among these endoplasmic reticulum- and Golgi-localized proteins requires Sed5p and is syntaxin-selective. In addition, Sed5p-containing SNARE complexes form selectively and this selectivity is mediated by Sed5p-containing intermediates that discriminate among subsequent binding partners. Although many of these SNAREs have overlapping distributions in vivo, the SNAREs that form complexes with Sed5p in vitro reflect their functionally distinct locales. Although SNARE-SNARE interactions are promiscuous and a single SNARE protein is often found in more than one complex, both the biochemical as well as genetic analyses reported here suggest that this is not a result of nonselective direct substitution of one SNARE for another. Rather the data are consistent with the existence of multiple (perhaps parallel) trafficking pathways where Sed5p-containing SNARE complexes play overlapping and/or distinct functional roles (Tsui, 2001).
The yeast SNARE Ykt6p has been implicated in several trafficking steps, including vesicular transport from the endoplasmic reticulum (ER) to the Golgi, intra-Golgi transport, and homotypic vacuole fusion. The functional role of its mammalian homolog (Ykt6) has not been established. Using antibodies specific for mammalian Ykt6, it is revealed that it is found mainly in Golgi-enriched membranes. Three SNAREs, syntaxin 5, GS28, and Bet1, are specifically associated with Ykt6 as revealed by co-immunoprecipitation, suggesting that these four SNAREs form a SNARE complex. Double labeling of Ykt6 and the Golgi marker mannosidase II or the ER-Golgi recycling marker KDEL receptor suggests that Ykt6 is primarily associated with the Golgi apparatus. Unlike the KDEL receptor, Ykt6 does not cycle back to the peripheral ER exit sites. Antibodies against Ykt6 inhibit in vitro ER-Golgi transport of vesicular stomatitis virus envelope glycoprotein (VSVG) only when they are added before the EGTA-sensitive stage. ER-Golgi transport of VSVG in vitro is also inhibited by recombinant Ykt6. In the presence of antibodies against Ykt6, VSVG accumulates in peri-Golgi vesicular structures and is prevented from entering the mannosidase II compartment, suggesting that Ykt6 functions at a late stage in ER-Golgi transport. Golgi apparatus marked by mannosidase II is fragmented into vesicular structures in cells microinjected with Ykt6 antibodies. It is concluded that Ykt6 functions in a late step of ER-Golgi transport, and this role may be important for the integrity of the Golgi complex (Zhang, 2001).
Syntaxin-5 (Sed5) is the only syntaxin needed for transport into and across the yeast Golgi, raising the question of how a single syntaxin species could mediate vesicle transport in both the anterograde and the retrograde direction within the stack. Sed5 is known to combine with two light chains (Bos1 and Sec22) to form the t-SNARE needed to receive vesicles from the endoplasmic reticulum. However, the yeast Golgi contains several other potential light chains with which Sed5 could potentially combine to form other t-SNAREs. To explore the degree of specificity in the choice of light chains by a t-SNARE, a comprehensive examination was undertaken of the capacity of all 21 Sed5-based t-SNAREs that theoretically could assemble in the yeast Golgi to fuse with each of the 7 potential v-SNAREs also present in this organelle. Only one additional of these 147 combinations was fusogenic. This functional proteomic strategy thereby revealed a previously uncharacterized t-SNARE in which Sed5 is the heavy chain and Gos1 and Ykt6 are the light chains, and whose unique cognate v-SNARE is Sft1. Immunoprecipitation experiments confirmed the existence of this complex in vivo. Fusion mediated by this second Golgi SNAREpin is topologically restricted, and existing genetic and morphologic evidence implies that it is used for transport across the Golgi stack. From this study, together with the previous functional proteomic analyses that have tested 275 distinct quaternary SNARE combinations, it follows that the fusion potential and transport pathways of the yeast cell can be read out from its genome sequence according to the SNARE hypothesis with a predictive accuracy of about 99.6% (Parlati, 2002).
Fusion of transport vesicles with their target organelles involves specific membrane proteins -- SNAREs -- which form tight complexes bridging the membranes to be fused. Evidence from yeast and mammals indicates that Sec1 family proteins act as regulators of membrane fusion by binding to the target membrane SNAREs. In experiments with purified proteins, the ER to Golgi core SNARE fusion complex can be assembled on syntaxin Sed5p tightly bound to the Sec1-related Sly1p. Sly1p also binds to preassembled SNARE complexes in vitro and is found to be part of a vesicular/target membrane SNARE complex immunoprecipitated from yeast cell lysates. This is in marked contrast to the exocytic SNARE assembly in neuronal cells where high affinity binding of N-Sec1/Munc-18 to syntaxin 1A precludes core SNARE fusion complex formation. The kinetics of SNARE complex formation in vitro with either Sly1p-bound or free Sed5p is not significantly different. Importantly, several presumably nonphysiological SNARE complexes easily generated with Sed5p do not form when the syntaxin is first bound to Sly1p. This indicates for the first time that a Sec1 family member contributes to the specificity of SNARE complex assembly (Peng, 2002).
Cytosolic Sec1/munc18-like proteins (SM proteins) are recruited to membrane fusion sites by interaction with syntaxin-type SNARE proteins, constituting indispensable positive regulators of intracellular membrane fusion. The crystal structure is presented of the yeast SM protein Sly1p in complex with a short N-terminal peptide derived from the Golgi-resident syntaxin Sed5p. Sly1p folds, similarly to neuronal Sec1, into a three-domain arch-shaped assembly, and Sed5p interacts in a helical conformation predominantly with domain I of Sly1p on the opposite site of the nSec1/syntaxin-1-binding site. Sequence conservation of the major interactions suggests that homologs of Sly1p as well as the paralogous Vps45p group bind their respective syntaxins in the same way. Furthermore, indirect evidence is presented that nSec1 might be able to contact syntaxin 1 in a similar fashion. The observed Sly1p-Sed5p interaction mode therefore indicates how SM proteins can stay associated with the assembling fusion machinery in order to participate in late fusion steps (Bracher, 2002).
SLY1 is an essential gene for vesicular transport between the ER and the early Golgi apparatus in Saccharomyces cerevisiae. It encodes a hydrophilic Sec1/Munc18 family protein that binds to the t-SNAREs. The amount of Sly1 protein that coprecipitates with the t-SNARE Sed5 is much reduced in a temperature-sensitive sly1(ts) mutant yeast compared with the wildtype. The mutant Sly1(ts) protein has a reduced binding activity to Sed5. In the wildtype, a detectable amount of Sly1 was found in the complex between Sed5 and the v-SNARE Bet1. In vitro formation of this complex on different membranes in yeast lysate is enhanced by the addition of recombinant Sly1. These results indicate that binding of Sly1 to Sed5 enhances trans-SNARE complex formation (Kosodo, 2002).
Syntaxins are a family of vesicular transport receptors that are involved in membrane traffic through both the constitutive and regulated secretory pathways. Syntaxins 1A/B,2,3, and 4 are principally associated with the plasma membrane. Two of the syntaxins, 1A and 1B, have been suggested to be the docking receptors for synaptic vesicles with the presynaptic membrane. The most distant member of the family, syntaxin 5, has been found in the Golgi region and has significant homology (35% identity) with Sed5p, an essential protein in yeast that is required for vesicular transport from the endoplasmic reticulum (ER) to the Golgi stack. Evidence that syntaxin 5 performs an analogous function in ER to Golgi transport in mammalian cells. Transient expression of an hemagglutinin-tagged full-length clone of syntaxin 5 and a truncated mutant lacking the transmembrane domain inhibits the transport of vesicular stomatitis virus glycoprotein to the Golgi stack. Under these conditions, vesicular stomatitis virus glycoprotein accumulates in pre-Golgi intermediates, which were strongly enriched in syntaxin 5. These results suggest that syntaxin 5 is the functional mammalian homologue of Sed5p and provides evidence for its role in regulating the potential targeting and/or fusion of carrier vesicles following export from the ER (Dascher, 1994).
A cell-free system that mimics the reassembly of Golgi stacks at the end of mitosis requires two ATPases, NSF and p97, to rebuild Golgi cisternae. Morphological studies now show that alpha-SNAP, a component of the NSF pathway, can inhibit the p97 pathway, whereas p47, a component of the p97 pathway, can inhibit the NSF pathway. Anti-syntaxin 5 antibodies and a soluble, recombinant syntaxin 5 inhibit both pathways, suggesting that this t-SNARE is a common component. Biochemical studies confirmed this, showing that p47 binds directly to syntaxin 5 and competes for binding with alpha-SNAP. p47 also mediates the binding of p97 to syntaxin 5 and so plays an analogous role to alpha-SNAP, which mediates the binding of NSF (Rabouille, 1998).
Syntaxins are thought to function during vesicular transport as receptors on the target membrane and to contribute to the specificity of membrane docking and fusion by interacting with vesicle-associated receptors. Syntaxin 5 (Syn5) has been shown to be an integral component of endoplasmic reticulum-derived transport vesicles. This pool, but not the target, Golgi-associated Syn5 pool, is essential for the assembly of vesicular-tubular pre-Golgi intermediates and the delivery of cargo to the Golgi. The requirement for vesicle-associated Syn5 in transport suggests a reevaluation of the basis for operation of the early secretory pathway (Rowe, 1998).
How do secretory proteins and other cargo targeted to post-Golgi locations traverse the Golgi stack? Immunoelectron microscopy experiments have established that a Golgi-restricted SNARE, GOS 28, is present in the same population of COPI vesicles as anterograde cargo marked by vesicular stomatitis virus glycoprotein, but is excluded from the COPI vesicles containing retrograde-targeted cargo (marked by KDEL receptor). GOS 28 and its partnering t-SNARE heavy chain, syntaxin 5, reside together in every cisterna of the stack. Taken together, these data raise the possibility that the anterograde cargo-laden COPI vesicles, retained locally by means of tethers, are inherently capable of fusing with neighboring cisternae on either side. If so, quanta of exported proteins would transit the stack in GOS 28-COPI vesicles via a bidirectional random walk, entering at the cis face and leaving at the trans face and percolating up and down the stack in between. Percolating vesicles carrying both post-Golgi cargo and Golgi residents up and down the stack would reconcile disparate observations on Golgi transport in cells and in cell-free systems (Orci, 2000).
The budding of vesicles from endoplasmic reticulum (ER) that contains nascent proteins is regulated by COPII proteins. The mechanisms that regulate lipid-carrying pre-chylomicron transport vesicles (PCTVs) budding from the ER are unknown. To study the dependence of PCTV-ER budding on COPII proteins protein and PCTV budding were examined by using ER prepared from rat small intestinal mucosal cells prelabeled with 3H-oleate or 14C-oleate and 3H-leucine. Budded 3H-oleate-containing PCTVs were separated by sucrose density centrifugation and were revealed by electron microscopy as 142-500 nm vesicles. The results show the following: (1) Proteinase K treatment does not degrade the PCTV cargo protein, apolipoprotein B-48, unless Triton X-100 is added; (2) PCTV budding is dependent on cytosol and ATP; (3) the COPII proteins Sar1, Sec24 and Sec13/31 and the membrane proteins syntaxin 5 and rBet1 are associated with PCTVs; (4) isolated PCTVs are able to fuse with intestinal Golgi; (5) antibodies to Sar1 completely inhibit protein vesicle budding but increase the generation of PCTV; these changes are reversed by the addition of recombinant Sar1; (6) PCTVs formed in the absence of Sar1 do not contain the COPII proteins Sar1, Sec24 or Sec31 and do not fuse with the Golgi complex. Together, these findings suggest that COPII proteins may not be required for the exit of membrane-bound chylomicrons from the ER but that they or other proteins may be necessary for PCTV fusion with the Golgi (Siddiqi, 2003).
Members of the syntaxin gene family are components of protein complexes that regulate vesicle docking and/or fusion during transport of cargo through the secretory pathway of eukaryotic cells. Syntaxin 5 is specifically required for endoplasmic reticulum to Golgi transport. A protein from rat liver membranes has been cloned that forms a native complex with syntaxin 5. This protein is the mammalian homolog to yeast Sly1p, previously identified as a protein that genetically and biochemically interacts with the small GTPase Ypt1p and Sed5p, proteins involved in docking/fusion in the early secretory pathway of yeast. Using transient expression it has been found that overexpression of rat liver Sly1 (rSly1) can neutralize the dominant negative effects of excess syntaxin 5 on endoplasmic reticulum to Golgi transport. These results suggest that rSly1 functions to positively regulate syntaxin 5 function (Dascher, 1996).
The proposed cis-Golgi vesicle receptor syntaxin 5 is found in a complex with Golgi-associated SNARE of 28 kDa (GOS-28), rbet1, rsly1, and two novel proteins characterized in this study: rat sec22b and membrin, both are cytoplasmically oriented integral membrane proteins. The complex appears to recapitulate vesicle docking interactions of proteins originating from distinct compartments, since syntaxin 5, rbet1, and GOS-28 localize to Golgi membranes, whereas mouse sec22b and membrin accumulate in the endoplasmic reticulum. Protein interactions in the complex are dramatically rearranged by N-ethylmaleimide-sensitive factor. The complex consists of two or more subcomplexes with some members (rat sec22b and syntaxin 5) in common and others (rbet1 and GOS-28) mutually exclusively associated. It is proposed that these protein interactions determine vesicle docking/fusion fidelity between the endoplasmic reticulum and Golgi (Hay, 1997).
SNAP receptor (SNARE) complexes bridge opposing membranes to promote membrane fusion within the secretory and endosomal pathways. Because only the exocytic SNARE complexes have been characterized in detail, the structural features shared by SNARE complexes from different fusion steps are not known. The subunit structure, assembly, and regulation is described of a quaternary SNARE complex, which appears to mediate an early step in endoplasmic reticulum (ER) to Golgi transport. Purified recombinant syntaxin 5, membrin, and rbet1, three Q-SNAREs, assemble cooperatively to create a high affinity binding site for sec22b, an R-SNARE. The syntaxin 5 amino-terminal domain potently inhibits SNARE complex assembly. The ER/Golgi quaternary complex is remarkably similar to the synaptic complex, suggesting that a common pattern is followed at all transport steps, where three Q-helices assemble to form a high affinity binding site for a fourth R-helix on an opposing membrane. Interestingly, although sec22b binds to the combination of syntaxin 5, membrin, and rbet1, it can only bind if it is present while the others assemble; sec22b cannot bind to a pre-assembled ternary complex of syntaxin 5, membrin, and rbet1. Finally, the quaternary complex containing sec22b is demonstrated to be not only an in vitro entity, but is a bona fide species in living cells as well (Xu, 2000).
p115 tethers coat protein (COP)I vesicles to Golgi membranes. The acidic COOH-terminal domain of p115 links the Golgins, Giantin on COPI vesicles, to GM130 on Golgi membranes. A SNARE motif-related domain within p115 stimulates the specific assembly of endogenous Golgi SNAREpins containing the t-SNARE, syntaxin 5. p115 catalyzes the construction of a cognate GOS-28-syntaxin-5 (v-/t-SNARE) complex by first linking the SNAREs to promote their direct interaction. These events are essential for NSF-catalyzed reassembly of postmitotic Golgi vesicles and tubules into mature cisternae. Staging experiments reveal that the linking of Golgins precedes SNAREpin assembly. Thus, p115 coordinates sequential tethering and docking of COPI vesicles by first using long tethers (Golgins) and then short tethers (SNAREs) (Shorter, 2002).
Characterization of mammalian NSF (G274E) and Drosophila NSF (comatose) mutants reveals an evolutionarily conserved NSF activity distinct from ATPase-dependent SNARE disassembly that is essential for Golgi membrane fusion. Analysis of mammalian NSF function during cell-free assembly of Golgi cisternae from mitotic Golgi fragments reveals that NSF disassembles Golgi SNAREs during mitotic Golgi fragmentation. A subsequent ATPase-independent NSF activity restricted to the reassembly phase is essential for membrane fusion. NSF/alpha-SNAP catalyzes the binding of GATE-16 to GOS-28, a Golgi v-SNARE, in a manner that requires ATP but not ATP hydrolysis. GATE-16 is essential for NSF-driven Golgi reassembly and precludes GOS-28 from binding to its cognate t-SNARE, syntaxin-5. It is suggested that this occurs at the inception of Golgi reassembly to protect the v-SNARE and regulate SNARE function (Muller, 2002).
Sec1/munc18-like proteins (SM proteins) and SNARE complexes are probably universally required for membrane fusion. However, the molecular mechanism by which they interact has only been defined for synaptic vesicle fusion where munc18 binds to syntaxin in a closed conformation that is incompatible with SNARE complex assembly. Sly1, an SM protein involved in Golgi and ER fusion, binds to a short, evolutionarily conserved N-terminal peptide of Sed5p and Ufe1p in yeast and of syntaxins 5 and 18 in vertebrates. In these syntaxins, the Sly1 binding peptide is upstream of a separate, autonomously folded N-terminal domain. These data suggest a potentially general mechanism by which SM proteins could interact with peptides in target proteins independent of core complex assembly and suggest that munc18 binding to syntaxin is an exception (Yamaguchi, 2002).
The subcellular localization, interacting partners, and function of GS15, a Golgi SNARE, remain to be established. Unlike proteins (Bet1 and the KDEL receptor) cycling between the Golgi and the intermediate compartment (IC, inclusive of the ER exit sites), GS15 is not redistributed into the IC upon incubation at 15 degrees C or when cells are treated with brefeldin A. Immuno-electron microscopy (immuno-EM) reveals that GS15 is mainly found in the medial-cisternae of the Golgi apparatus and adjacent tubulo-vesicular elements. Coimmunoprecipitation experiments suggest that GS15 exists in a distinct SNARE complex that contains SNAREs (syntaxin5, GS28, and Ykt6) that are implicated in both ER-to-Golgi and intra-Golgi transport but not with SNAREs involved exclusively in ER-to-Golgi traffic. Furthermore, components of COPI coat can be selectively coimmunoprecipitated with GS15 from Golgi extracts. Overexpression of mutant forms of GS15 affects the normal distribution of cis- and medial-Golgi proteins (GS28, syntaxin 5, and Golgi mannosidase II), whereas proteins of the trans-Golgi and TGN (Vti1-rp2/Vti1a and syntaxin 6) and Golgi matrix/scaffold (GM130 and p115) are less affected. When the level of GS15 is reduced by duplex 21-nt small interfering RNA (siRNA)-mediated knockdown approach, diverse markers of the Golgi apparatus are redistributed into small dotty and diffuse labeling, suggesting an essential role of GS15 in the Golgi apparatus (Y. Xu, 2002).
Sec1Munc18-like (SM) proteins functionally interact with soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNARE) in membrane fusion, but the mechanisms of these interactions differ. In vertebrates, SM proteins that mediate exocytosis (Munc18-1, 18-2, and 18c) bind to the closed conformation of syntaxins 1-4, which requires the N-terminal H(abc) domains and SNARE motifs of these syntaxins. In contrast, SM proteins that mediate Golgi and endoplasmic reticulum fusion (Sly1 and Vps45) bind only to short N-terminal sequences of syntaxins 5, 16, or 18, independently of their H(abc) domains and SNARE motifs. Munc18-1, Sly1, and Vps45 interact with cognate syntaxins via similar, autonomously folded N-terminal domains, but the syntaxin 5-binding surface of the Sly1 N-terminal domain is opposite the syntaxin 1-binding surface of the Munc18-1 N-terminal domain. In transfected cells, the N-terminal domain of Sly1 specifically disrupts the structure of the Golgi complex, supporting the notion that the interaction of Sly1 with syntaxin 5 is essential for fusion. These data, together with previous results, suggest that a relatively small N-terminal domain of SM proteins is dedicated to mechanistically distinct interactions with SNAREs, leaving the remaining large parts of SM proteins free to execute their as yet unknown function as effector domains (Dulubova, 2003).
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date revised: 22 November 2022
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