Jesse C Hay

University of Montana, MSO, Montana, United States

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Publications (27)253.8 Total impact

  • Ting Wang · Robert Grabski · Elizabeth Sztul · Jesse C. Hay
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    ABSTRACT: Tethering factors regulate the targeting of membrane-enclosed vesicles under the control of Rab GTPases. P115, a golgin family tether, has been shown to participate in multiple stages of ER/Golgi transport. Despite extensive study, p115's mechanism of action is poorly understood. SNARE proteins make up the machinery for membrane fusion, and strong evidence shows that p115's function is directly linked to its interaction with SNAREs. Using a gel filtration binding assay, we have demonstrated that in solution p115 stably interacts with ER/Golgi SNAREs rbet1 and sec22b, but not membrin and syntaxin 5. These binding preferences stemmed from selectivity of p115 for monomeric SNARE motifs as opposed to SNARE oligomers. Soluble monomeric rbet1 can compete off p115 from COPII vesicles. Furthermore, excess p115 inhibits p115 function in trafficking. We conclude that monomeric SNAREs are a major binding site for p115 on COPII vesicles, and that p115 dissociates from its SNARE partners upon SNAREpin assembly. Our results suggest a model in which p115 forms a mixed p115/SNARE helix bundle with a monomeric SNARE, facilitates the binding activity and/or concentration of the SNARE at pre-fusion sites, and is subsequently ejected as SNARE complex formation and fusion proceed.
    Traffic 11/2014; 16(2). DOI:10.1111/tra.12242 · 4.71 Impact Factor
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    ABSTRACT: Luminal calcium released from secretory organelles has been suggested to play a regulatory role in vesicle transport at several steps in the secretory pathway, however, its functional roles and effector pathways have not been elucidated. Here we demonstrate for the first time that specific luminal calcium depletion leads to a significant decrease in ER-to-Golgi transport rates in intact cells. Ultrastructural analysis revealed that luminal calcium depletion is accompanied by increased accumulation of intermediate compartment (IC) proteins in COPII buds and clusters of unfused COPII vesicles at ER exit sites (ERES). Furthermore, we present several lines of evidence suggesting that luminal calcium affected transport at least in part through calcium-dependent interactions between apoptosis linked gene-2 (ALG-2) and the sec31A proline-rich region (PRR): 1) targeted disruption of ALG-2/sec31A interactions caused severe defects in ER-to-Golgi transport in intact cells; 2) effects of luminal calcium and ALG-2/sec31A interactions on transport mutually required each other, and; 3) sec31A function in transport required luminal calcium. Morphological phenotypes of disrupted ALG-2/sec31A interactions were characterized. We found that ALG-2/sec31A interactions were not required for the localization of sec31A to ERES per se, but appeared to acutely regulate the stability and trafficking of the cargo receptor p24 and the distribution of the vesicle tether protein p115. These results represent the first outline of a mechanism that connects luminal calcium to specific protein interactions regulating vesicle trafficking machinery.
    Journal of Biological Chemistry 07/2014; 289(34). DOI:10.1074/jbc.M114.561829 · 4.57 Impact Factor
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    ABSTRACT: Traffic from the ER to Golgi complex is initiated when the activated form of the GTPase Sar1p recruits the Sec23p/Sec24p complex to ER membranes. The Sec23p/Sec24p complex, which forms the inner shell of the COPII coat, sorts cargo into ER-derived vesicles. The coat inner shell recruits the Sec13p/Sec31p complex, leading to coat polymerization and vesicle budding. Recent studies have revealed that the Sec23p subunit sequentially interacts with three different binding partners to direct a COPII vesicle to the Golgi. One of these binding partners is the serine/threonine kinase Hrr25p. Hrr25p phosphorylates the COPII coat, driving the membrane-bound pool into the cytosol. The phosphorylated coat cannot rebind to the ER to initiate a new round of vesicle budding unless it is dephosphorylated. Here we screened all the known protein phosphatases in yeast to identify one whose loss of function alters the cellular distribution of COPII coat subunits. This screen identified the PP2A-like phosphatase Sit4p as a regulator of COPII coat dephosphorylation. Hyperphosphorylated coat subunits accumulate in the sit4Δ mutant in vivo. In vitro, Sit4p dephosphorylates COPII coat subunits. Consistent with a role in coat recycling, Sit4p and its mammalian ortholog, PP6, regulate traffic from the ER to the Golgi complex.
    Molecular biology of the cell 07/2013; 24(17). DOI:10.1091/mbc.E13-02-0114 · 5.98 Impact Factor
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    ABSTRACT: The membrane tethering factor p115 has been shown to have important functions in ER to Golgi traffic and Golgi biogenesis. The multidomain structure of p115 allows for interactions with a diverse array of proteins that govern cargo movement at the ER-Golgi interface. Within its C-terminal region p115 contains four coiled-coil domains (CC1-CC4). Of the four coiled-coils, only CC1 has been shown to be required for p115 function, presumably by its ability to bind numerous SNARE proteins as well as the small GTPase Rab1. Recently, we showed that CC4 also interacts with SNARE proteins and that CC4 is required for p115 function in Golgi homeostasis and the trafficking of transmembrane but not soluble cargo. Here, we propose a novel model wherein p115 facilitates membrane tethering and fusion by simultaneously engaging its CC1 and CC4 domains with distinct SNARE proteins to promote formation of SNARE complexes.
    Bioarchitecture 09/2012; 2(5). DOI:10.4161/bioa.21702
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    Meg Trahey · Hyung Suk Oh · Craig E Cameron · Jesse C Hay
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    ABSTRACT: Poliovirus (PV) requires membranes of the host cell's secretory pathway to generate replication complexes (RCs) for viral RNA synthesis. Recent work identified the intermediate compartment and the Golgi apparatus as the precursors of the replication "organelles" of PV (N. Y. Hsu et al., Cell 141:799-811, 2010). In this study, we examined the effect of PV on COPII vesicles, the secretory cargo carriers that bud from the endoplasmic reticulum and homotypically fuse to form the intermediate compartment that matures into the Golgi apparatus. We found that infection by PV results in a biphasic change in functional COPII vesicle biogenesis in cells, with an early enhancement and subsequent inhibition. Concomitant with the early increase in COPII vesicle formation, we found an increase in the membrane fraction of Sec16A, a key regulator of COPII vesicle formation. We suggest that the early burst in COPII vesicle formation detected benefits PV by increasing the precursor pool required for the formation of its RCs.
    Journal of Virology 06/2012; 86(18):9675-82. DOI:10.1128/JVI.01159-12 · 4.65 Impact Factor
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    ABSTRACT: The arginine-type soluble N-ethylmaleimide-sensitive factor attachment protein receptor (R-SNARE) ykt6 possesses several atypical properties including selective high expression in neurons, a lipidated C-terminus, localization to punctae that do not correspond with known endomembrane markers, a potent ability to protect the secretory pathway from alpha-synuclein over-expression and specific up-regulation in tumors. We have followed up on several of these features that together suggest nontraditional SNARE structures and functions. A significant portion of ykt6 in PC12 cells was found in a protease-resistant state suggestive of a large complex or aggregate. Other endoplasmic reticulum/Golgi SNAREs were not protease resistant, demonstrating that SNARE complexes per se did not cause protease resistance. Mutagenesis indicated that lipidation of the ykt6 C-terminus was also not involved, implicating its longin domain in particle formation. Immunogold electron microscopy revealed ykt6 labeling of ∼100 nm electron densities associated with diverse membranes. Density gradient analysis of the protease-resistant structures confirmed their tight association with membranes. Since excess ykt6 has been correlated with tumorigenesis, we tested whether ykt6 over-expression in normal rat kidney cells that normally express little ykt6 affected the cell cycle. Ykt6 over-expression was found to result in altered cell division cycles as evidenced by significantly smaller cells, a higher mitotic index and increased DNA synthesis. Mutagenesis studies dis-correlated SNARE function with the cell cycle effects; instead, the cell cycle effects correlated better with ykt6 properties related to the longin domain or particle formation. The ykt6 particles/aggregates may represent ykt6 engaged in a non-SNARE function(s) or else nonfunctional, stored and/or excess ykt6. Whether the particulate ykt6 structures represent a means of buffering the apparent proliferative activity or are in fact mechanistically related to this activity will be of future interest in neuroscience and cancer biology.
    Biology of the Cell 03/2012; 104(7):397-417. DOI:10.1111/boc.201100048 · 3.87 Impact Factor
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    ABSTRACT: How the directionality of vesicle traffic is achieved remains an important unanswered question in cell biology. The Sec23p/Sec24p coat complex sorts the fusion machinery (SNAREs) into vesicles as they bud from the endoplasmic reticulum (ER). Vesicle tethering to the Golgi begins when the tethering factor TRAPPI binds to Sec23p. Where the coat is released and how this event relates to membrane fusion is unknown. Here we use a yeast transport assay to demonstrate that an ER-derived vesicle retains its coat until it reaches the Golgi. A Golgi-associated kinase, Hrr25p (CK1δ orthologue), then phosphorylates the Sec23p/Sec24p complex. Coat phosphorylation and dephosphorylation are needed for vesicle fusion and budding, respectively. Additionally, we show that Sec23p interacts in a sequential manner with different binding partners, including TRAPPI and Hrr25p, to ensure the directionality of ER-Golgi traffic and prevent the back-fusion of a COPII vesicle with the ER. These events are conserved in mammalian cells.
    Nature 05/2011; 473(7346):181-6. DOI:10.1038/nature09969 · 42.35 Impact Factor
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    Meg Trahey · Jesse C Hay
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    ABSTRACT: Transport vesicle coat proteins play active roles in vesicle cargo sorting as well as membrane deformation and fission during vesicle biogenesis. For years, it was assumed that this was the extent of the coats' function and that the coats depolymerized immediately after vesicle budding, leaving the exposed fusion machinery free to find, dock, and fuse with the proper target membrane. Recently, however, it has become increasingly clear that the coat remains on transport vesicles during their post-budding life and in fact helps properly pair up the vesicle with its intended target membrane. These data have brought up urgent questions about exactly when vesicles do uncoat and how uncoating is regulated. Here, we summarize the latest round of evidence for post-budding roles for coats, including a few hints about how the uncoating process may be coupled to docking and fusion. We also speculate about the possibility of post-fusion functions for residual coats.
    F1000 Biology Reports 06/2010; 2:47. DOI:10.3410/B2-47
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    ABSTRACT: Toxicity of human alpha-synuclein when expressed in simple organisms can be suppressed by overexpression of endoplasmic reticulum (ER)-to-Golgi transport machinery, suggesting that inhibition of constitutive secretion represents a fundamental cause of the toxicity. Whether similar inhibition in mammals represents a cause of familial Parkinson's disease has not been established. We tested elements of this hypothesis by expressing human alpha-synuclein in mammalian kidney and neuroendocrine cells and assessing ER-to-Golgi transport. Overexpression of wild type or the familial disease-associated A53T mutant alpha-synuclein delayed transport by up to 50%; however, A53T inhibited more potently. The secretory delay occurred at low expression levels and was not accompanied by insoluble alpha-synuclein aggregates or mistargeting of transport machinery, suggesting a direct action of soluble alpha-synuclein on trafficking proteins. Co-overexpression of ER/Golgi arginine soluble N-ethylmaleimide-sensitive factor attachment protein receptors (R-SNAREs) specifically rescued transport, indicating that alpha-synuclein antagonizes SNARE function. Ykt6 reversed alpha-synuclein inhibition much more effectively than sec22b, suggesting a possible neuroprotective role for the enigmatic high expression of ykt6 in neurons. In in vitro reconstitutions, purified alpha-synuclein A53T protein specifically inhibited COPII vesicle docking and fusion at a pre-Golgi step. Finally, soluble alpha-synuclein A53T directly bound ER/Golgi SNAREs and inhibited SNARE complex assembly, providing a potential mechanism for toxic effects in the early secretory pathway.
    Molecular biology of the cell 06/2010; 21(11):1850-63. DOI:10.1091/mbc.E09-09-0801 · 5.98 Impact Factor
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    ABSTRACT: The significance and extent of Ca(2+) regulation of the biosynthetic secretory pathway have been difficult to establish, and our knowledge of regulatory relationships integrating Ca(2+) with vesicle coats and function is rudimentary. Here, we investigated potential roles and mechanisms of luminal Ca(2+) in the early secretory pathway. Specific depletion of luminal Ca(2+) in living normal rat kidney cells using cyclopiazonic acid (CPA) resulted in the extreme expansion of vesicular tubular cluster (VTC) elements. Consistent with this, a suppressive role for vesicle-associated Ca(2+) in COPII vesicle homotypic fusion was demonstrated in vitro using Ca(2+) chelators. The EF-hand-containing protein apoptosis-linked gene 2 (ALG-2), previously implicated in the stabilization of sec31 at endoplasmic reticulum exit sites, inhibited COPII vesicle fusion in a Ca(2+)-requiring manner, suggesting that ALG-2 may be a sensor for the effects of vesicular Ca(2+) on homotypic fusion. Immunoisolation established that Ca(2+) chelation inhibits and ALG-2 specifically favors residual retention of the COPII outer shell protein sec31 on pre-Golgi fusion intermediates. We conclude that vesicle-associated Ca(2+), acting through ALG-2, favors the retention of residual coat molecules that seem to suppress membrane fusion. We propose that in cells, these Ca(2+)-dependent mechanisms temporally regulate COPII vesicle interactions, VTC biogenesis, cargo sorting, and VTC maturation.
    Molecular biology of the cell 03/2010; 21(6):1033-46. DOI:10.1091/mbc.E09-10-0914 · 5.98 Impact Factor
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    Jesse C Hay
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    ABSTRACT: For many years, it has been known that an increase in cytosolic calcium triggers the fusion of secretory granules and synaptic vesicles with the plasma membrane. However, the role of calcium in the intracellular membrane-fusion reactions that coordinate the secretory and endocytic pathways has been less clear. Initially, there was accumulating evidence to indicate that a focally localized and transient calcium signal is required to trigger even those fusion events formerly classified as 'constitutive'-that is, those that normally occur in the absence of global cytosolic calcium increases. Therefore, calcium seemed to be a required fundamental co-factor underlying all biological membrane-fusion steps, perhaps with a conserved mechanism of action. However, although such unification would be gratifying, new data indicate that several intracellular fusion events do not require calcium after all. In this review, the evidence for calcium requirements and its modes of action in constitutive trafficking are discussed. As a challenging perspective, I suggest that the specific absence of calcium requirements for some transport steps in fact expands the function of calcium in trafficking, because divergent luminal calcium concentrations and requirements for fusion might increase the specificity with which intracellular membrane-fusion partners are determined.
    EMBO Reports 04/2007; 8(3):236-40. DOI:10.1038/sj.embor.7400921 · 7.86 Impact Factor
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    ABSTRACT: The budding of endoplasmic reticulum (ER)-derived vesicles is dependent on the COPII coat complex. Coat assembly is initiated when Sar1-GTP recruits the cargo adaptor complex, Sec23/Sec24, by binding to its GTPase-activating protein (GAP) Sec23 (ref. 2). This leads to the capture of transmembrane cargo by Sec24 (refs 3, 4) before the coat is polymerized by the Sec13/Sec31 complex. The initial interaction of a vesicle with its target membrane is mediated by tethers. We report here that in yeast and mammalian cells the tethering complex TRAPPI (ref. 7) binds to the coat subunit Sec23. This event requires the Bet3 subunit. In vitro studies demonstrate that the interaction between Sec23 and Bet3 targets TRAPPI to COPII vesicles to mediate vesicle tethering. We propose that the binding of TRAPPI to Sec23 marks a coated vesicle for fusion with another COPII vesicle or the Golgi apparatus. An implication of these findings is that the intracellular destination of a transport vesicle may be determined in part by its coat and its associated cargo.
    Nature 03/2007; 445(7130):941-4. DOI:10.1038/nature05527 · 42.35 Impact Factor
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    ABSTRACT: In mammals, coat complex II (COPII)-coated transport vesicles deliver secretory cargo to vesicular tubular clusters (VTCs) that facilitate cargo sorting and transport to the Golgi. We documented in vitro tethering and SNARE-dependent homotypic fusion of endoplasmic reticulum-derived COPII transport vesicles to form larger cargo containers characteristic of VTCs ( Xu, D., and Hay, J. C. (2004) J. Cell Biol. 167, 997-1003). COPII vesicles thus appear to contain all necessary components for homotypic tethering and fusion, providing a pathway for de novo VTC biogenesis. Here we demonstrate that antibodies against the endoplasmic reticulum/Golgi SNARE Syntaxin 5 inhibit COPII vesicle homotypic tethering as well as fusion, implying an unanticipated role for SNAREs upstream of fusion. Inhibition of SNARE complex access and/or disassembly with dominant-negative alpha-soluble NSF attachment protein (SNAP) also inhibited tethering, implicating SNARE status as a critical determinant in COPII vesicle tethering. The tethering-defective vesicles generated in the presence of dominant-negative alpha-SNAP specifically lacked the Rab1 effectors p115 and GM130 but not other peripheral membrane proteins. Furthermore, Rab effectors, including p115, were shown to be required for homotypic COPII vesicle tethering. Thus, our results demonstrate a requirement for SNARE-dependent tether recruitment and function in COPII vesicle fusion. We anticipate that recruitment of tether molecules by an upstream SNARE signal ensures that tethering events are initiated only at focal sites containing appropriately poised fusion machinery.
    Journal of Biological Chemistry 01/2007; 281(50):38825-33. DOI:10.1074/jbc.M606044200 · 4.57 Impact Factor
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    ABSTRACT: TRAPPI is a large complex that mediates the tethering of COPII vesicles to the Golgi (heterotypic tethering) in the yeast Saccharomyces cerevisiae. In mammalian cells, COPII vesicles derived from the transitional endoplasmic reticulum (tER) do not tether directly to the Golgi, instead, they appear to tether to each other (homotypic tethering) to form vesicular tubular clusters (VTCs). We show that mammalian Bet3p (mBet3p), which is the most highly conserved TRAPP subunit, resides on the tER and adjacent VTCs. The inactivation of mBet3p results in the accumulation of cargo in membranes that colocalize with the COPII coat. Furthermore, using an assay that reconstitutes VTC biogenesis in vitro, we demonstrate that mBet3p is required for the tethering and fusion of COPII vesicles to each other. Consistent with the proposal that mBet3p is required for VTC biogenesis, we find that ERGIC-53 (VTC marker) and Golgi architecture are disrupted in siRNA-treated mBet3p-depleted cells. These findings imply that the TRAPPI complex is essential for VTC biogenesis.
    The Journal of Cell Biology 08/2006; 174(3):359-68. DOI:10.1083/jcb.200603044 · 9.69 Impact Factor
  • Ashwini P Joglekar · Jesse C Hay
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    ABSTRACT: SNARE proteins control intracellular membrane fusion through formation of membrane-bridging helix bundles of amphipathic SNARE motifs. Repetitive cycles of membrane fusion likely involve repetitive folding/unfolding of the SNARE motif helical structure. Despite these conformational demands, little is known about conformational regulation of SNAREs by other proteins. Here we demonstrate that hsc70 chaperones stimulate in vitro SNARE complex formation among the ER/Golgi SNAREs syntaxin 5, membrin, rbetl and sec22b, under conditions in which assembly is normally inhibited. Thus, molecular chaperones can render the SNARE motif more competent for assembly. Partially purified hsc70 fractions from brain cytosol had higher specific activities than fully purified hsc70, suggesting the involvement of unidentified cofactors. Using chemical crosslinking of cells followed by immunoprecipitation, we found that hsc70 was associated with ER/Golgi SNAREs in vivo. Consistent with a modulatory role for hsc70 in transport, we found that excess hsc70 specifically inhibited ER-to-Golgi transport in permeabilized cells.
    European Journal of Cell Biology 07/2005; 84(5):529-42. DOI:10.1016/j.ejcb.2004.12.028 · 3.70 Impact Factor
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    ABSTRACT: Arf and Rab family GTPases regulate membrane traffic in cells, yet little is known about how they are targeted to distinct organelles. To identify sequences in Arf-1 necessary for Golgi targeting, we examined the localization of chimeras between Arf-1 and Arf-6. Here, we identify a 16-amino acid sequence in Arf-1 that specifies Golgi targeting and contains a motif (MXXE) that is important for Arf-1 binding to membrin, an ER-Golgi SNARE protein. The MXXE motif is conserved in all Arfs known to localize to the Golgi and enables Arf-1 to localize to the early Golgi. Arf-1 lacking these 16 aa can still localize to the late Golgi where it displays a more rapid Golgi-cytosol cycle than wild-type Arf-1. These studies suggest that membrin recruits Arf-1 to the early Golgi and reveal distinct kinetic cycles for Arf-1 at early and late Golgi determined by different sets of Arf regulators and effectors.
    The Journal of Cell Biology 04/2005; 168(7):1039-51. DOI:10.1083/jcb.200409138 · 9.69 Impact Factor
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    Dalu Xu · Jesse C Hay
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    ABSTRACT: What is the first membrane fusion step in the secretory pathway? In mammals, transport vesicles coated with coat complex (COP) II deliver secretory cargo to vesicular tubular clusters (VTCs) that ferry cargo from endoplasmic reticulum exit sites to the Golgi stack. However, the precise origin of VTCs and the membrane fusion step(s) involved have remained experimentally intractable. Here, we document in vitro direct tethering and SNARE-dependent fusion of endoplasmic reticulum-derived COPII transport vesicles to form larger cargo containers. The assembly did not require detectable Golgi membranes, preexisting VTCs, or COPI function. Therefore, COPII vesicles appear to contain all of the machinery to initiate VTC biogenesis via homotypic fusion. However, COPI function enhanced VTC assembly, and early VTCs acquired specific Golgi components by heterotypic fusion with Golgi-derived COPI vesicles.
    The Journal of Cell Biology 01/2005; 167(6):997-1003. DOI:10.1083/jcb.200408135 · 9.69 Impact Factor
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    ABSTRACT: Although the membrane-trafficking functions of most SNAREs are conserved from yeast to humans, some mammalian SNAREs have evolved specialized functions unique to multicellular life. The mammalian homolog of the prenylated yeast SNARE Ykt6p might be one such example, because rat Ykt6 is highly expressed only in brain neurons. Furthermore, neuronal Ykt6 displayed a remarkably specialized, punctate localization that did not overlap appreciably with conventional compartments of the endomembrane system, suggesting that Ykt6 might be involved in a pathway unique to or specifically modified for neuronal function. Targeting of Ykt6 to its unique subcellular location was directed by its profilin-like longin domain. We have taken advantage of high-resolution structural data available for the yeast Ykt6p longin domain to examine mechanisms by which the mammalian longin domain controls Ykt6 conformation and subcellular targeting. We found that the overall tertiary structure of the longin domain, not sequence-specific surface features, drives direct targeting to the Ykt6 punctate structures. However, several sequence-specific surface features of the longin domain indirectly regulate Ykt6 localization through intramolecular interactions that mask otherwise-dominant targeting signals on the SNARE motif and lipid groups. Specifically, two hydrophobic binding pockets, one on each face of the longin domain, and one mixed hydrophobic/charged surface, participate in protein-protein interactions with the SNARE motif and protein-lipid interactions with the lipid group(s) at the molecule's C-terminus. One of the hydrophobic pockets suppresses protein-palmitoylation-dependent mislocalization of Ykt6 to the plasma membrane. The Ykt6 intramolecular interactions would be predicted to create a compact, closed conformation of the SNARE that prevents promiscuous targeting interactions and premature insertion into membranes. Interestingly, both protein-protein and protein-lipid interactions are required for a tightly closed conformation and normal targeting.
    Journal of Cell Science 10/2004; 117(Pt 19):4495-508. DOI:10.1242/jcs.01314 · 5.33 Impact Factor
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    ABSTRACT: Although some of the principles of N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) function are well understood, remarkably little detail is known about sec1/munc18 (SM) protein function and its relationship to SNAREs. Popular models of SM protein function hold that these proteins promote or maintain an open and/or monomeric pool of syntaxin molecules available for SNARE complex formation. To address the functional relationship of the mammalian endoplasmic reticulum/Golgi SM protein rsly1 and its SNARE binding partner syntaxin 5, we produced a conformation-specific monoclonal antibody that binds only the available, but not the cis-SNARE-complexed nor intramolecularly closed form of syntaxin 5. Immunostaining experiments demonstrated that syntaxin 5 SNARE motif availability is nonuniformly distributed and focally regulated. In vitro endoplasmic reticulum-to-Golgi transport assays revealed that rsly1 was acutely required for transport, and that binding to syntaxin 5 was absolutely required for its function. Finally, manipulation of rsly1-syntaxin 5 interactions in vivo revealed that they had remarkably little impact on the pool of available syntaxin 5 SNARE motif. Our results argue that although rsly1 does not seem to regulate the availability of syntaxin 5, its function is intimately associated with syntaxin binding, perhaps promoting a later step in SNARE complex formation or function.
    Molecular Biology of the Cell 02/2004; 15(1):162-75. DOI:10.1091/mbc.E03-07-0535 · 4.55 Impact Factor
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    ABSTRACT: The endoplasmic reticulum/Golgi SNARE rbet1 cycles between the endoplasmic reticulum and Golgi and is essential for cargo transport in the secretory pathway. Although the quaternary SNARE complex containing rbet1 is known to function in membrane fusion, the structural role of rbet1 is unclear. Furthermore, the structural determinants for rbet1 targeting and its cyclical itinerary have not been investigated. We utilized protein interaction assays to demonstrate that the rbet1 SNARE motif plays a structural role similar to the carboxyl-terminal helix of SNAP-25 in the synaptic SNARE complex and demonstrated the importance to SNARE complex assembly of a conserved salt bridge between rbet1 and sec22b. We also examined the potential role of the rbet1 SNARE motif and SNARE interactions in rbet1 localization and dynamics. We found that, in contrast to what has been observed for syntaxin 5, the rbet1 SNARE motif was essential for proper targeting. To test whether SNARE interactions were important for the targeting function of the SNARE motif, we used charge repulsion mutations at the conserved salt bridge position that rendered rbet1 defective for binary, ternary, and quaternary SNARE interactions. We found that heteromeric SNARE interactions are not required at any step in rbet1 targeting or dynamics. Furthermore, the heteromeric state of the SNARE motif does not influence its interaction with the COPI coat or efficient recruitment onto transport vesicles. We conclude that protein targeting is a completely independent function of the rbet1 SNARE motif, which is capable of distinct classes of protein interactions.
    Journal of Biological Chemistry 05/2003; 278(16):14121-33. DOI:10.1074/jbc.M300659200 · 4.57 Impact Factor

Publication Stats

1k Citations
253.80 Total Impact Points

Institutions

  • 2005–2014
    • University of Montana
      • • Division of Biological Sciences
      • • Center for Structural and Functional Neuroscience
      MSO, Montana, United States
  • 2012
    • Pennsylvania State University
      • Department of Biochemistry and Molecular Biology
      University Park, Maryland, United States
  • 2010
    • Medical University of Graz
      Gratz, Styria, Austria
  • 2007
    • University of Alabama at Birmingham
      Birmingham, Alabama, United States
  • 1998–2007
    • Howard Hughes Medical Institute
      Ashburn, Virginia, United States
  • 2001–2005
    • University of Michigan
      • Department of Molecular, Cellular and Developmental Biology
      Ann Arbor, Michigan, United States