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Γ-SNAP Stimulates Disassembly of Endosomal SNARE Complexes and Regulates Endocytic Trafficking Pathways

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Γ-SNAP Stimulates Disassembly of Endosomal SNARE Complexes and Regulates Endocytic Trafficking Pathways © 2015. Published by The Company of Biologists Ltd | Journal of Cell Science (2015) 128, 2781-2794 doi:10.1242/jcs.158634 RESEARCH ARTICLE γ-SNAP stimulates disassembly of endosomal SNARE complexes and regulates endocytic trafficking pathways Hiroki Inoue1,*, Yuka Matsuzaki1, Ayaka Tanaka1, Kaori Hosoi1, Kaoru Ichimura2, Kohei Arasaki1, Yuichi Wakana1, Kenichi Asano1, Masato Tanaka1, Daisuke Okuzaki3, Akitsugu Yamamoto2, Katsuko Tani1 and Mitsuo Tagaya1,* ABSTRACT specific organelles and transport vesicles, and mediates membrane Soluble N-ethylmaleimide-sensitive factor attachment protein transport in specific endocytic and exocytic routes. For example, receptors (SNAREs) that reside in the target membranes and SNAREs such as syntaxin (STX)4, STX7 and STX18 catalyze transport vesicles assemble into specific SNARE complexes to membrane fusion in the plasma membrane, endosomes and drive membrane fusion. N-ethylmaleimide-sensitive factor (NSF) and lysosomes, and the endoplasmic reticulum (ER), respectively its attachment protein, α-SNAP (encoded by NAPA), catalyze (Hatsuzawa et al., 2000; Sumitani et al., 1995; Wong et al., 1998). disassembly of the SNARE complexes in the secretory and SNARE proteins are classified into four groups, Qa, Qb, Qc and R, endocytic pathways to recycle them for the next round of fusion according to the sequence similarity of the SNARE motif and its events. γ-SNAP (encoded by NAPG) is a SNAP isoform, but its flanking regions (Hong, 2005; Jahn and Scheller, 2006). In most function in SNARE-mediated membrane trafficking remains cases, Qa-, Qb- and Qc-SNAREs reside in target membranes and thus unknown. Here, we show that γ-SNAP regulates the endosomal are also called target (t)-SNAREs. By contrast, most R-SNAREs trafficking of epidermal growth factor (EGF) receptor (EGFR) and reside in transport vesicles and thus are also called vesicle (v)- transferrin. Immunoprecipitation and mass spectrometry analyses SNAREs. Three Q-SNAREs and one R-SNARE form a specific four- revealed that γ-SNAP interacts with a limited range of SNAREs, helical bundle complex between opposing membranes of organelles including endosomal ones. γ-SNAP, as well as α-SNAP, mediated and transport vesicles in trans, and drive membrane fusion. The the disassembly of endosomal syntaxin-7-containing SNARE SNARE complex that is formed in a membrane as a result of the complexes. Overexpression and small interfering (si)RNA-mediated fusion event is disassembled and recycled for the next reaction by γ ATPase N-ethylmaleimide sensitive factor (NSF) and its attachment depletion of -SNAP changed the morphologies and intracellular α α distributions of endosomes. Moreover, the depletion partially protein, soluble NSF-attachment protein ( -SNAP; encoded by NAPA). Structural and biochemical analyses have revealed that three suppressed the exit of EGFR and transferrin from EEA1-positive α early endosomes to delay their degradation and uptake. Taken -SNAPs bind to hetero-tetramer SNARE complex and recruit the together, our findings suggest that γ-SNAP is a unique SNAP that NSF hexamer (Antonin et al., 2002; Chang et al., 2012; Söllner et al., – – 1993; Stein et al., 2009; Wimmer et al., 2001). functions in a limited range of organelles including endosomes α β and their trafficking pathways. In mammals, there are three SNAP isoforms: -SNAP, -SNAP (encoded by NAPB)andγ-SNAP (encoded by NAPG). In contrast to KEY WORDS: NAPG, STX7, STX8, Syntaxin 8, Endocytosis ubiquitous expression of α-SNAP, β-SNAP is specifically expressed in brain (Whiteheart et al., 1993). These two isoforms share more INTRODUCTION than 80% amino acid sequence identity, and thus β-SNAP can also Membrane trafficking in eukaryotic cells plays pivotal roles in a bind to and catalyze the disassembly of the SNARE complex, and the wide variety of cellular functions. Transport vesicles containing isoforms act together in regulated exocytosis in neuronal cells cargo molecules – e.g. secretory and membrane proteins – are (Sudlow et al., 1996; Xu et al., 2002). γ-SNAP is as ubiquitously generated from donor membranes, and they are then transported to, expressed as α-SNAP and is only ∼25% identical in its amino acid and become tethered to and fused with target membranes through sequence to both α-SNAP and β-SNAP. In contrast to the numerous cellular machineries. Soluble N-ethylmaleimide sensitive considerable contribution of α-SNAP and β-SNAP to SNARE factor attachment protein receptors (SNAREs) are key elements of complexes in membrane trafficking events, the biochemical and the membrane fusion machinery. At least 38 genes encoding cellular functions of γ-SNAP remain poorly understood. Yeast two- SNAREs exist in the human genome, and all of them have one or hybrid screening experiments have revealed that γ-SNAP interacts two insertions of a characteristic α-helical sequence called the with γ-SNAP-associated factor-1 [Gaf-1; also known as Rab11- SNARE motif (Hong, 2005; Hong and Lev, 2014; Jahn and interacting protein (Rip11) and Rab11-family interacting protein 5 Scheller, 2006). Each SNARE is preferentially distributed in (FIP5)], γ-tubulin and NSF (Chen et al., 2001; Tani et al., 2003). The two-hybrid assay also revealed that γ-SNAP does not bind to at least to six of the SNAREs tested, including STX4 and STX18, although 1 School of Life Sciences, Tokyo University of Pharmacy and Life Sciences, Hachioji, α-SNAP does bind to them (Tani et al., 2003). Moreover, γ-SNAP is Tokyo 192-0392, Japan. 2Faculty of Bioscience, Nagahama Institute of Bio-Science and Technology, Nagahama, Shiga 526-0829, Japan. 3Department of Molecular not required for vesicle-mediated transport of vesicular stomatitis Genetics, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka virus G protein (VSVG) from the ER to the Golgi, but α-SNAP is 565-0871, Japan. essential for this process (Peter et al., 1998). Subramaniam et al., *Authors for correspondence ([email protected]; [email protected]) however, have shown that the endosomal SNARE STX8 is associated with not only α-SNAP and NSF, but also γ-SNAP, as revealed by Received 24 June 2014; Accepted 17 June 2015 immunoprecipitation of STX8 (Subramaniam et al., 2000). Journal of Cell Science 2781 RESEARCH ARTICLE Journal of Cell Science (2015) 128, 2781-2794 doi:10.1242/jcs.158634 In this study, to determine the biochemical and cellular functions with the SNARE domain of STX8, as α-SNAP did (supplementary of γ-SNAP, we sought to identify binding partners of γ-SNAP by material Fig. S1C). Moreover, the binding of γ-SNAP to STX7 using immunoprecipitation and mass spectrometry analyses. Here, could be reduced upon competition with an excess amount of α- we show that γ-SNAP binds to selected SNAREs and catalyzes the SNAP (supplementary material Fig. S1D,E). These results suggest disassembly of the endosomal STX7–STX8 SNARE complex, but that γ-SNAP directly binds to STX proteins and that it has the same not of the Golgi-associated STX5–Bet1 SNARE complex. We also binding site on SNAREs as α-SNAP. observed that overexpression and small interfering (si)RNA- We next sought to define the residues and/or domain that mediated depletion of γ-SNAP affects the morphology and determine the preference of the interaction of γ-SNAP for intracellular localization of endosomes. Moreover, the depletion endosomal SNAREs. First, we used two chimeras of α-SNAP and delayed the trafficking of EGFR and transferrin (Tfn) from early γ-SNAP to probe their interactions with endogenous STX4 (plasma endosomes, but did not affect the constitutive secretion of membrane SNARE) and STX8 (endosomal SNARE) horseradish peroxidase fused with a secretory signal sequence (supplementary material Fig. S1F). A chimera comprising the N- (ssHRP). Our findings suggest that γ-SNAP promotes endosomal terminal half of bovine α-SNAP (amino acid residues 1–164) and SNARE disassembly and that it is required for normal endosome the C-terminal half of bovine γ-SNAP (amino acid residues 157– function. 312), termed NαCγ, bound well to STX8, as the wild-type proteins do, whereas the chimera still bound to STX4, but the binding was RESULTS much weaker than that of wild-type α-SNAP. By contrast, another γ-SNAP preferentially binds to endosomal SNAREs chimera NγCα, comprising the N-terminal half of γ-SNAP (amino To gain new insights into the function of γ-SNAP, we sought to acid residues 1–156) and the C-terminal half of α-SNAP (amino identify γ-SNAP-interacting proteins by immunoprecipitation and acid residues 165–295) bound to neither STX4 nor STX8. These mass spectrometry analyses. Triple-FLAG-tagged γ-SNAP results suggest that α-SNAP primarily binds to STX4 through its N- (3×FLAG–γ-SNAP) was transiently expressed in 293T cells and terminal half, as proposed previously (Marz et al., 2003; Rice and immunoprecipitated. The precipitated proteins were separated by Brunger, 1999), but that its C-terminal half also makes some using SDS-PAGE, silver-stained and identified mass spectrometry contribution to the binding and that the C-terminal half of γ-SNAP (Fig. 1A,B). In addition to NSF, which we have previously reported plays a more important role in its binding to STX8. (Tani et al., 2003), several SNARE proteins, SNARE-associated Marz et al. (2003) have proposed that eight positively charged proteins (Sly1/Sec1-family domain containing proteins 1 and 2, residues in the concave surface of the N-terminal twisted-sheet SCFD1 and SCFD2, respectively), α-SNAP and other membrane- domain of α-SNAP electrostatically interact with a cluster of trafficking-related proteins – including clathrin heavy chain and negatively charged residues in SNARE complexes that comprise phosphoinositide phosphatase Sac1 – were detected. Among these, STX1. To define the residues that determine the binding preference, we decided to focus on SNAREs because their interactions with γ- we focused on two – Lys94 and Lys163 – because their SNAP have remained poorly characterized (Chen et al., 2001; corresponding residues in γ-SNAP are negatively charged residues Hong, 2005; Tani et al., 2003).
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