Class C Vps protein complex regulates vacuolar SNARE pairing and is required for vesicle docking/fusion

In yeast, the Class C Vps protein complex (C-Vps complex), composed of Vps11, Vps16, Vps18, and Vps33, functions in Golgi-to-vacuole protein transport. In this study, we characterized and purified this complex and identified its interaction with the syntaxin homolog Vam3. Vam3 pairs with the SNAP-25 homolog Vam7 and VAMP homolog Vti1 to form SNARE complexes during vesicle docking/fusion with the vacuole. The C-Vps complex does not bind to Vam3-Vti1-Vam7 paired SNARE complexes but instead binds to unpaired Vam3. Antibodies to a component of this complex inhibited in vitro vacuole-to-vacuole fusion. Furthermore, temperature-conditional mutations in the Class C VPS genes destabilized Vam3-Vti1-Vam7 pairing. Therefore, we propose that the C-Vps complex associates with unpaired (activated) Vam3 to mediate the assembly of trans-SNARE complexes during both vesicle docking/fusion and vacuole-to-vacuole fusion.     

How Tlg2p/syntaxin 16 'snares' Vps45

Soluble N-ethylmaleimide sensitive factor-attachment protein receptors (SNAREs) and Sec1p/Munc18-homologs (SM proteins) play key roles in intracellular membrane fusion. The SNAREs form tight four-helix bundles (core complexes) that bring the membranes together, but it is unclear how this activity is coupled to SM protein function. Studies of the yeast trans-Golgi network (TGN)/endosomal SNARE complex, which includes the syntaxin-like SNARE Tlg2p, have suggested that its assembly requires activation by binding of the SM protein Vps45p to the cytoplasmic region of Tlg2p folded into a closed conformation. Nuclear magnetic resonance and biochemical experiments now show that Tlg2p and Pep12p, a late- endosomal syntaxin that interacts functionally but not directly with Vps45p, have a domain structure characteristic of syntaxins but do not adopt a closed conformation. Tlg2p binds tightly to Vps45p via a short N-terminal peptide motif that is absent in Pep12p. The Tlg2p/Vps45p binding mode is shared by the mammalian syntaxin 16, confirming that it is a Tlg2p homolog, and resembles the mode of interaction between the SM protein Sly1p and the syntaxins Ufe1p and Sed5p. Thus, this mechanism represents the most widespread mode of coupling between syntaxins and SM proteins.     

The Sec1p/Munc18 protein Vps45p binds its cognate SNARE proteins via two distinct modes

Sec1p/Munc18 (SM) proteins are essential for SNARE-mediated membrane trafficking. The formulation of unifying hypotheses for the function of the SM protein family has been hampered by the observation that two of its members bind their cognate syntaxins (Sxs) in strikingly different ways. The SM protein Vps45p binds its Sx Tlg2p in a manner analogous to that captured by the Sly1p-Sed5p crystal structure, whereby the NH2-terminal peptide of the Sx inserts into a hydrophobic pocket on the outer face of domain I of the SM protein. In this study, we report that although this mode of interaction is critical for the binding of Vps45p to Tlg2p, the SM protein also binds Tlg2p-containing SNARE complexes via a second mode that involves neither the NH2 terminus of Tlg2p nor the region of Vps45p that facilitates this interaction. Our findings point to the possibility that SM proteins interact with their cognate SNARE proteins through distinct mechanisms at different stages in the SNARE assembly/disassembly cycle.     

Vps45p stabilizes the syntaxin homologue Tlg2p and positively regulates SNARE complex formation.

Sec1p-like/Munc-18 (SM) proteins bind to t-SNAREs and inhibit ternary complex formation. Paradoxically, the absence of SM proteins does not result in constitutive membrane fusion. Here, we show that in yeast cells lacking the SM protein Vps45p, the t-SNARE Tlg2p is down-regulated, to undetectable levels, by rapid proteasomal degradation. In the absence of Vps45p, Tlg2p can be stabilized through abolition of proteasome activity. Surprisingly, the stabilized Tlg2p was targeted to the correct intracellular location. However, the stabilized Tlg2p is non-functional and unable to bind its cognate SNARE binding partners, Tlg1p and Vti1p, in the absence of Vps45p. A truncation mutant lacking the first 230 residues of Tlg2p no longer bound Vps45p but was able to form complexes with Tlg1p and Vti1p in the absence of the SM protein. These data provide us with two valuable insights into the function of SM proteins. First, SM proteins act as chaperone-like molecules for their cognate t-SNAREs. Secondly, SM proteins play an essential role in the activation process allowing their cognate t-SNARE to participate in ternary complex formation.     

Sec18p and Vam7p remodel trans-SNARE complexes to permit a lipid-anchored R-SNARE to support yeast vacuole fusion

Intracellular membrane fusion requires SNARE proteins in a trans-complex, anchored to apposed membranes. Proteoliposome studies have suggested that SNAREs drive fusion by stressing the lipid bilayer via their transmembrane domains (TMDs), and that SNARE complexes require a TMD in each docked membrane to promote fusion. Yeast vacuole fusion is believed to require three Q-SNAREs from one vacuole and the R-SNARE Nyv1p from its fusion partner. In accord with this model, we find that fusion is abolished when the TMD of Nyv1p is replaced by lipid anchors, even though lipid-anchored Nyv1p assembles into trans-SNARE complexes. However, normal fusion is restored by the addition of both Sec18p and the soluble SNARE Vam7p. In restoring fusion, Sec18p promotes the disassembly of trans-SNARE complexes, and Vam7p enhances their assembly. Thus, either the TMD of this R-SNARE is not essential for fusion, and TMD-mediated membrane stress is not the only mode of trans-SNARE complex action, or these SNAREs have more flexibility than heretofore appreciated to form alternate functional complexes that violate the 3Q:1R rule.     

Cellular levels of the syntaxin Tlg2p are regulated by a single mode of binding to Vps45p

Sec1p/Munc18 (SM) proteins play a key role in the regulation of soluble N-ethylmaleimide-sensitive fusion (NSF)-attachment protein receptor (SNARE)-mediated intracellular membrane trafficking events in all eukaryotic cells. Understanding the molecular mechanisms by which SM proteins function has not been straight forward as SM proteins bind to their cognate SNARE proteins by at least two distinct mechanisms, suggesting that they provide more than one function. We have previously characterised two binding modes used by the yeast SM protein Vps45p to interact with its SNARE proteins. In one of these modes, the N terminus of the syntaxin Tlg2p inserts into a hydrophobic pocket in the SM protein. We now report that disruption of this high-affinity binding between Vps45p and Tlg2p leads to downregulation of Tlg2p, and propose that this pocket-mode of binding of SM proteins to their cognate syntaxins serves to regulate cellular levels of the syntaxin.     

The Phosphatidylinositol 3-Phosphate Binding Protein Vac1p Interacts with a Rab GTPase and a Sec1p Homologue to Facilitate Vesicle-mediated Vacuolar Protein Sorting

Activated GTP-bound Rab proteins are thought to interact with effectors to elicit vesicle targeting and fusion events. Vesicle-associated v-SNARE and target membrane t-SNARE proteins are also involved in vesicular transport. Little is known about the functional relationship between Rabs and SNARE protein complexes. We have constructed an activated allele of VPS21, a yeast Rab protein involved in vacuolar protein sorting, and demonstrated an allele-specific interaction between Vps21p and Vac1p. Vac1p was found to bind the Sec1p homologue Vps45p. Although no association between Vps21p and Vps45p was seen, a genetic interaction between VPS21 and VPS45 was observed. Vac1p contains a zinc-binding FYVE finger that may bind phosphatidylinositol 3-phosphate [PtdIns(3)P]. In other FYVE domain proteins, this motif and PtdIns(3)P are necessary for membrane association. Vac1 proteins with mutant FYVE fingers still associated with membranes but showed vacuolar protein sorting defects and reduced interactions with Vps45p and activated Vps21p. Vac1p membrane association was not dependent on PtdIns(3)P, Pep12p, Vps21p, Vps45p, or the PtdIns 3-kinase, Vps34p. Vac1p FYVE finger mutant missorting phenotypes were suppressed by a defective allele of VPS34. These data indicate that PtdIns(3)P may perform a regulatory role, possibly involved in mediating Vac1p protein-protein interactions. We propose that activated-Vps21p interacts with its effector, Vac1p, which interacts with Vps45p to regulate the Golgi to endosome SNARE complex.     

An InCytes from MBC Selection: Yeast Sec1p Functions before and after Vesicle Docking

Sec1/Munc18 (SM) proteins bind cognate soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes and stimulate vesicle membrane fusion. Before fusion, vesicles are docked to specific target membranes. Regulation of vesicle docking is attributed to some but not all SM proteins, suggesting specialization of this earlier function. Yeast Sec1p seems to function only after vesicles are docked and SNARE complexes are assembled. Here, we show that yeast Sec1p is required before and after SNARE complex assembly, in support of general requirements for SM proteins in both vesicle docking and fusion. Two classes of sec1 mutants were isolated. Class A mutants are tightly blocked in cell growth and secretion at a step before SNARE complex assembly. Class B mutants have a SNARE complex binding defect, with a range in severity of cell growth and secretion defects. Mapping the mutations onto an SM protein structure implicates a peripheral bundle of helices for the early, docking function and a deep groove, opposite the syntaxin-binding cleft on nSec1/Munc-18, for the interaction between Sec1p and the exocytic SNARE complex.     

A soluble SNARE drives rapid docking, bypassing ATP and Sec17/18p for vacuole fusion

Membrane fusion requires priming, the disassembly of cis-SNARE complexes by the ATP-driven chaperones Sec18/17p. Yeast vacuole priming releases Vam7p, a soluble SNARE. Vam7p reassociation during docking allows trans-SNARE pairing and fusion. We now report that recombinant Vam7p (rVam7p) enters into complex with other SNAREs in vitro and bypasses the need for Sec17p, Sec18p, and ATP. Thus, the sole essential function of vacuole priming in vitro is the release of Vam7p from cis-SNARE complexes. In 'bypass fusion', without ATP but with added rVam7p, there are sufficient unpaired vacuolar SNAREs Vam3p, Vti1p, and Nyv1p to interact with Vam7p and support fusion. However, active SNARE proteins are not sufficient for bypass fusion. rVam7p does not bypass requirements for Rho GTPases,Vps33p, Vps39p, Vps41p, calmodulin, specific lipids, or Vph1p, a subunit of the V-ATPase. With excess rVam7p, reduced levels of PI(3)P or functional Ypt7p suffice for bypass fusion. High concentrations of rVam7p allow the R-SNARE Ykt6p to substitute for Nyv1p for fusion; this functional redundancy among vacuole SNAREs may explain why nyv1delta strains lack the vacuole fragmentation seen with mutants in other fusion catalysts.     

The Sec1/Munc18 protein, Vps33p, functions at the endosome and the vacuole of Saccharomyces cerevisiae

The Sec1/Munc18 (SM) family of proteins is thought to impart compartmental specificity to vesicle fusion reactions. Here we report characterization of Vps33p, an SM family member previously thought to act exclusively at the vacuolar membrane with the vacuolar syntaxin Vam3p. Vacuolar morphology of vps33Delta cells resembles that of cells lacking both Vam3p and the endosomal syntaxin Pep12p, suggesting that Vps33p may function with these syntaxins at the vacuole and the endosome. Consistent with this, vps33 mutants secrete the Golgi precursor form of the vacuolar hydrolase CPY into the medium. We also demonstrate that Vps33p acts at other steps, for vps33 mutants show severe defects in endocytosis at the late endosome. At the endosome, Vps33p and other class C members exist as a complex with Vps8p, a protein previously known to act in transport between the late Golgi and the endosome. Vps33p also interacts with Pep12p, a known interactor of the SM protein Vps45p. High copy PEP7/VAC1 suppresses vacuolar morphology defects of vps33 mutants. These findings demonstrate that Vps33p functions at multiple trafficking steps and is not limited to action at the vacuolar membrane. This is the first report demonstrating the involvement of a single syntaxin with two SM proteins at the same organelle.     

HOPS proofreads the trans-SNARE complex for yeast vacuole fusion

The fusion of yeast vacuoles, like other organelles, requires a Rab-family guanosine triphosphatase (Ypt7p), a Rab effector and Sec1/Munc18 (SM) complex termed HOPS (homotypic fusion and vacuole protein sorting), and soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs). The central 0-layer of the four bundled vacuolar SNAREs requires the wild-type three glutaminyl (Q) and one arginyl (R) residues for optimal fusion. Alterations of this layer dramatically increase the K(m) value for SNAREs to assemble trans-SNARE complexes and to fuse. We now find that added purified HOPS complex strongly suppresses the fusion of vacuoles bearing 0-layer alterations, but it has little effect on the fusion of vacuoles with wild-type SNAREs. HOPS proofreads at two levels, inhibiting the formation of trans-SNARE complexes with altered 0-layers and suppressing the ability of these mismatched 0-layer trans-SNARE complexes to support membrane fusion. HOPS proofreading also extends to other parts of the SNARE complex, because it suppresses the fusion of trans-SNARE complexes formed without the N-terminal Phox homology domain of Vam7p (Q(c)). Unlike some other SM proteins, HOPS proofreading does not require the Vam3p (Q(a)) N-terminal domain. HOPS thus proofreads SNARE domain and N-terminal domain structures and regulates the fusion capacity of trans-SNARE complexes, only allowing full function for wild-type SNARE configurations. This is the most direct evidence to date that HOPS is directly involved in the fusion event.     

Structural analysis of the interaction between the SNARE Tlg1 and Vps51

Membrane fusion in cells involves the interaction of SNARE proteins on apposing membranes. Formation of SNARE complexes is preceded by tethering events, and a number of protein complexes that are thought to mediate this have been identified. The VFT or GARP complex is required for endosome-Golgi traffic in yeast. It consists of four subunits, one of which, Vps51, has been shown to bind specifically to the SNARE Tlg1, which participates in the same fusion event. We have determined the structure of the N-terminal domain of Tlg1 bound to a peptide from the N terminus of Vps51. Binding depends mainly on residues 18-30 of Vps51. These form a short helix which lies in a conserved groove in the three-helix bundle formed by Tlg1. Surprisingly, although both Vps51 and Tlg1 are required for transport to the late Golgi from endosomes, removal of the Tlg1-binding sequences from Vps51 does not block such traffic in vivo. Thus, this particular interaction cannot be crucial to the process of vesicle docking or fusion.     

Interaction of SNAREs with ArfGAPs precedes recruitment of Sec18p/NSF

Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins are key components of the fusion machinery in vesicular transport and in homotypic membrane fusion. We previously found that ADP-ribosylation factor GTPase activating proteins (ArfGAPs) promoted a conformational change on SNAREs that allowed recruitment of the small GTPase Arf1p in stoichiometric amounts. Here, we show that the ArfGAP Gcs1p accelerates vesicle (v)-target membrane (t)-SNARE complex formation in vitro, indicating that ArfGAPs may act as folding chaperones. These SNARE complexes were resolved in the presence of ATP by the yeast homologues of alpha-soluble N-ethylmaleimide-sensitive factor attachment protein and N-ethylmaleimide-sensitive factor, Sec17p and Sec18p, respectively. In addition, Sec18p and Sec17p also recognized the "activated" SNAREs even when they were not engaged in v-t-SNARE complexes. Here again, the induction of a conformational change by ArfGAPs was essential. Surprisingly, recruitment of Sec18p to SNAREs did not require Sec17p or ATP hydrolysis. Moreover, Sec18p displaced prebound Arf1p from SNAREs, indicating that Sec18p may have more than one function: first, to ensure that all vesicle coat proteins are removed from the SNAREs before the engagement in a trans-SNARE complex; and second, to resolve cis-SNARE complexes after fusion has occurred.     

Sec1/Munc18 protein Vps33 binds to SNARE domains and the quaternary SNARE complex

Soluble N-ethylmaleimide–sensitive factor attachment protein receptor (SNARE) proteins catalyze membrane fusion events in the secretory and endolysosomal systems, and all SNARE-mediated fusion processes require cofactors of the Sec1/Munc18 (SM) family. Vps33 is an SM protein and subunit of the Vps-C complexes HOPS (homotypic fusion and protein sorting) and CORVET (class C core vacuole/endosome tethering), which are central regulators of endocytic traffic. Here we present biochemical studies of interactions between Saccharomyces cerevisiae vacuolar SNAREs and the HOPS holocomplex or Vps33 alone. HOPS binds the N-terminal H_abc domain of the Qa-family SNARE Vam3, but Vps33 is not required for this interaction. Instead, Vps33 binds the SNARE domains of Vam3, Vam7, and Nyv1. Vps33 directly binds vacuolar quaternary SNARE complexes, and the affinity of Vps33 for SNARE complexes is greater than for individual SNAREs. Through targeted mutational analyses, we identify missense mutations of Vps33 that produce a novel set of defects, including cargo missorting and the loss of Vps33-HOPS association. Together these data suggest a working model for membrane docking: HOPS associates with N-terminal domains of Vam3 and Vam7 through Vps33-independent interactions, which are followed by binding of Vps33, the HOPS SM protein, to SNARE domains and finally to the quaternary SNARE complex. Our results also strengthen the hypothesis that SNARE complex binding is a core attribute of SM protein function.     

Sec1/Munc18 protein stabilizes fusion-competent syntaxin for membrane fusion in Arabidopsis cytokinesis

Intracellular membrane fusion requires complexes of syntaxins with other SNARE proteins and regulatory Sec1/Munc18 (SM) proteins. In membrane fusion mediating, e.g., neurotransmitter release or glucose-stimulated insulin secretion in mammals, SM proteins preferentially interact with the inactive closed, rather than the active open, conformation of syntaxin or with the assembled SNARE complex. Other membrane fusion processes such as vacuolar fusion in yeast involve like membranes carrying cis-SNARE complexes, and the role of SM protein is unknown. We investigated syntaxin-SM protein interaction in membrane fusion of Arabidopsis cytokinesis, which involves cytokinesis-specific syntaxin KNOLLE and SM protein KEULE. KEULE interacted with an open conformation of KNOLLE that complemented both knolle and keule mutants. This interaction occurred at the cell division plane and required the KNOLLE linker sequence between helix Hc and SNARE domain. Our results suggest that in cytokinesis, SM protein stabilizes the fusion-competent open form of syntaxin, thereby promoting trans-SNARE complex formation.     

Vps51p mediates the association of the GARP (Vps52/53/54) complex with the late Golgi t-SNARE Tlg1p

Multisubunit tethering complexes may contribute to the specificity of membrane fusion events by linking transport vesicles to their target membrane in an initial recognition event that promotes SNARE assembly. However, the interactions that link tethering factors to the other components of the vesicle fusion machinery are still largely unknown. We have previously identified three subunits of a Golgi-localized complex (the Vps52/53/54 complex) that is required for retrograde transport to the late Golgi. This complex interacts with a Rab and a SNARE protein found at the late Golgi and is related to two other multisubunit tethering complexes: the COG complex and the exocyst. Here we show that the Vps52/53/54 complex has an additional subunit, Vps51p. All four members of this tetrameric GARP (Golgi-associated retrograde protein) complex are required for two distinct retrograde transport pathways, from both early and late endosomes, back to the TGN. vps51 mutants exhibit a distinct phenotype suggestive of a regulatory role. Indeed, we find that Vps51p mediates the interaction between Vps52/53/54 and the t-SNARE Tlg1p. The binding of this small, coiled-coil protein to the conserved N-terminal domain of the t-SNARE therefore provides a crucial link between components of the tethering and the fusion machinery.     

Sec17p and HOPS, in distinct SNARE complexes, mediate SNARE complex disruption or assembly for fusion

SNARE functions during membrane docking and fusion are regulated by Sec1/Munc18 (SM) chaperones and Rab/Ypt GTPase effectors. These functions for yeast vacuole fusion are combined in the six-subunit HOPS complex. HOPS facilitates Ypt7p nucleotide exchange, is a Ypt7p effector, and contains an SM protein. We have dissected the associations and requirements for HOPS, Ypt7p, and Sec17/18p during SNARE complex assembly. Vacuole SNARE complexes bind either Sec17p or the HOPS complex, but not both. Sec17p and its co-chaperone Sec18p disassemble SNARE complexes. Ypt7p regulates the reassembly of unpaired SNAREs with each other and with HOPS, forming HOPS.SNARE complexes prior to fusion. After HOPS.SNARE assembly, lipid rearrangements are still required for vacuole content mixing. Thus, Sec17p and HOPS have mutually exclusive interactions with vacuole SNAREs to mediate disruption of SNARE complexes or their assembly for docking and fusion. Sec17p may displace HOPS from SNAREs to permit subsequent rounds of fusion.     

The Sec1/Munc18 Protein Vps45 Regulates Cellular Levels of Its SNARE Binding Partners Tlg2 and Snc2 in Saccharomyces cerevisiae

Intracellular membrane trafficking pathways must be tightly regulated to ensure proper functioning of all eukaryotic cells. Central to membrane trafficking is the formation of specific SNARE (soluble N-ethylmeleimide-sensitive factor attachment protein receptor) complexes between proteins on opposing lipid bilayers. The Sec1/Munc18 (SM) family of proteins play an essential role in SNARE-mediated membrane fusion, and like the SNAREs are conserved through evolution from yeast to humans. The SM protein Vps45 is required for the formation of yeast endosomal SNARE complexes and is thus essential for traffic through the endosomal system. Here we report that, in addition to its role in regulating SNARE complex assembly, Vps45 regulates cellular levels of its SNARE binding partners: the syntaxin Tlg2 and the v-SNARE Snc2: Cells lacking Vps45 have reduced cellular levels of Tlg2 and Snc2; and elevation of Vps45 levels results in concomitant increases in the levels of both Tlg2 and Snc2. As well as regulating traffic through the endosomal system, the Snc v-SNAREs are also required for exocytosis. Unlike most vps mutants, cells lacking Vps45 display multiple growth phenotypes. Here we report that these can be reversed by selectively restoring Snc2 levels in vps45 mutant cells. Our data indicate that as well as functioning as part of the machinery that controls SNARE complex assembly, Vps45 also plays a key role in determining the levels of its cognate SNARE proteins; another key factor in regulation of membrane traffic.     

Vac1p coordinates Rab and phosphatidylinositol 3-kinase signaling in Vps45p-dependent vesicle docking/fusion at the endosome

The vacuolar protein sorting (VPS) pathway of Saccharomyces cerevisiae mediates transport of vacuolar protein precursors from the late Golgi to the lysosome-like vacuole. Sorting of some vacuolar proteins occurs via a prevacuolar endosomal compartment and mutations in a subset of VPS genes (the class D VPS genes) interfere with the Golgi-to-endosome transport step. Several of the encoded proteins, including Pep12p/Vps6p (an endosomal target (t) SNARE) and Vps45p (a Sec1p homologue), bind each other directly [1]. Another of these proteins, Vac1p/Pep7p/Vps19p, associates with Pep12p and binds phosphatidylinositol 3-phosphate (PI(3)P), the product of the Vps34 phosphatidylinositol 3-kinase (PI 3-kinase) [1] [2]. Here, we demonstrate that Vac1p genetically and physically interacts with the activated, GTP-bound form of Vps21p, a Rab GTPase that functions in Golgi-to-endosome transport, and with Vps45p. These results implicate Vac1p as an effector of Vps21p and as a novel Sec1p-family-binding protein. We suggest that Vac1p functions as a multivalent adaptor protein that ensures the high fidelity of vesicle docking and fusion by integrating both phosphoinositide (Vps34p) and GTPase (Vps21p) signals, which are essential for Pep12p- and Vps45p-dependent targeting of Golgi-derived vesicles to the prevacuolar endosome.     

Crystal Structures of the Sec1/Munc18 (SM) Protein Vps33, Alone and Bound to the Homotypic Fusion and Vacuolar Protein Sorting (HOPS) Subunit Vps16*

Intracellular membrane fusion requires the regulated assembly of SNARE (soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptor) proteins anchored in the apposed membranes. To exert the force required to drive fusion between lipid bilayers, juxtamembrane SNARE motifs zipper into four-helix bundles. Importantly, SNARE function is regulated by additional factors, none more extensively studied than the SM (Sec1/Munc18-like) proteins. SM proteins interact with both individual SNAREs and SNARE complexes, likely chaperoning SNARE complex formation and protecting assembly intermediates from premature disassembly by NSF. Four families of SM proteins have been identified, and representative members of two of these families (Sec1/Munc18 and Sly1) have been structurally characterized. We report here the 2.6 Å resolution crystal structure of an SM protein from the third family, Vps33. Although Vps33 shares with the first two families the same basic three-domain architecture, domain 1 is displaced by 15 Å, accompanied by a 40° rotation. A unique feature of the Vps33 family of SM proteins is that its members function as stable subunits within a multi-subunit tethering complex called HOPS (homotypic fusion and vacuolar protein sorting). Integration into the HOPS complex depends on the interaction between Vps33 and a second HOPS subunit, Vps16. The crystal structure of Vps33 bound to a C-terminal portion of Vps16, also at 2.6 Å resolution, reveals the structural basis for this interaction. Despite the extensive interface between the two HOPS subunits, the conformation of Vps33 is only subtly affected by binding to Vps16.