Structural basis for endosomal recruitment of ESCRT-I by ESCRT-0 in yeast

The ESCRT‐0 and ESCRT‐I complexes coordinate the clustering of ubiquitinated cargo with intralumenal budding of the endosomal membrane, two essential steps in vacuolar/lysosomal protein sorting from yeast to humans. The 1.85‐Å crystal structure of interacting regions of the yeast ESCRT‐0 and ESCRT‐I complexes reveals that PSDP motifs of the Vps27 ESCRT‐0 subunit bind to a novel electropositive N‐terminal site on the UEV domain of the ESCRT‐I subunit Vps23 centred on Trp16. This novel site is completely different from the C‐terminal part of the human UEV domain that binds to P(S/T)AP motifs of human ESCRT‐0 and HIV‐1 Gag. Disruption of the novel PSDP‐binding site eliminates the interaction in vitro and blocks enrichment of Vps23 in endosome‐related class E compartments in yeast cells. However, this site is non‐essential for sorting of the ESCRT cargo Cps1. Taken together, these results show how a conserved motif/domain pair can evolve to use strikingly different binding modes in different organisms.     

The Linker Region Plays a Regulatory Role in Assembly and Activity of the Vps4 AAA ATPase

The AAA-type ATPase Vps4 functions with components of the ESCRT (endosomal sorting complex required for transport) machinery in membrane fission events that are essential for endosomal maturation, cytokinesis, and the formation of retroviruses. A key step in these events is the assembly of monomeric Vps4 into the active ATPase complex, which is aided in part by binding of Vps4 via its N-terminal MIT (microtubule interacting and trafficking) domain to its substrate ESCRT-III. We found that the 40-amino acid linker region between the MIT and the ATPase domain of Vps4 is not required for proper function but plays a role in regulating Vps4 assembly and ATPase activity. Deletion of the linker is expected to bring the MIT domains into close proximity to the central pore of the Vps4 complex. We propose that this localization of the MIT domain in linker-deleted Vps4 mimics a repositioning of the MIT domain normally caused by binding of Vps4 to ESCRT-III. This structure would allow the Vps4 complex to engage ESCRT-III subunits with both the pore and the MIT domain simultaneously, which might be essential for the ATP-driven disassembly of ESCRT-III.     

Binding of Substrates to the Central Pore of the Vps4 ATPase Is Autoinhibited by the Microtubule Interacting and Trafficking (MIT) Domain and Activated by MIT Interacting Motifs (MIMs)

The endosomal sorting complexes required for transport (ESCRT) pathway drives reverse topology membrane fission events within multiple cellular pathways, including cytokinesis, multivesicular body biogenesis, repair of the plasma membrane, nuclear membrane vesicle formation, and HIV budding. The AAA ATPase Vps4 is recruited to membrane necks shortly before fission, where it catalyzes disassembly of the ESCRT-III lattice. The N-terminal Vps4 microtubule-interacting and trafficking (MIT) domains initially bind the C-terminal MIT-interacting motifs (MIMs) of ESCRT-III subunits, but it is unclear how the enzyme then remodels these substrates in response to ATP hydrolysis. Here, we report quantitative binding studies that demonstrate that residues from helix 5 of the Vps2p subunit of ESCRT-III bind to the central pore of an asymmetric Vps4p hexamer in a manner that is dependent upon the presence of flexible nucleotide analogs that can mimic multiple states in the ATP hydrolysis cycle. We also find that substrate engagement is autoinhibited by the Vps4p MIT domain and that this inhibition is relieved by binding of either Type 1 or Type 2 MIM elements, which bind the Vps4p MIT domain through different interfaces. These observations support the model that Vps4 substrates are initially recruited by an MIM-MIT interaction that activates the Vps4 central pore to engage substrates and generate force, thereby triggering ESCRT-III disassembly.     

Biochemical Analyses of Human IST1 and Its Function in Cytokinesis

The newly described yeast endosomal sorting complexes required for transport (ESCRT) protein increased sodium tolerance-1 (Ist1p) binds the late-acting ESCRT proteins Did2p/charged MVB protein (CHMP) 1 and Vps4p and exhibits synthetic vacuolar protein sorting defects when combined with mutations in the Vta1p/LIP5–Vps60p/CHMP5 complex. Here, we report that human IST1 also functions in the ESCRT pathway and is required for efficient abscission during HeLa cell cytokinesis. IST1 binding interactions with VPS4, CHMP1, LIP5, and ESCRT-I were characterized, and the IST1–VPS4 interaction was investigated in detail. Mutational and NMR spectroscopic studies revealed that the IST1 terminus contains two distinct MIT interacting motifs (MIM1 and MIM2) that wrap around and bind in different groves of the MIT helical bundle. IST1, CHMP1, and VPS4 were recruited to the midbodies of dividing cells, and depleting either IST1 or CHMP1 proteins blocked VPS4 recruitment and abscission. In contrast, IST1 depletion did not inhibit human immunodeficiency virus-1 budding. Thus, IST1 and CHMP1 act together to recruit and modulate specific VPS4 activities required during the final stages of cell division.     

Structural studies of phosphoinositide 3-kinase-dependent traffic to multivesicular bodies

Three large protein complexes known as ESCRT I, ESCRT II and ESCRT III drive the progression of ubiquitinated membrane cargo from early endosomes to lysosomes. Several steps in this process critically depend on PtdIns3P, the product of the class III phosphoinositide 3-kinase. Our work has provided insights into the architecture, membrane recruitment and functional interactions of the ESCRT machinery. The fan-shaped ESCRT I core and the trilobal ESCRT II core are essential to forming stable, rigid scaffolds that support additional, flexibly-linked domains, which serve as gripping tools for recognizing elements of the MVB (multivesicular body) pathway: cargo protein, membranes and other MVB proteins. With these additional (non-core) domains, ESCRT I grasps monoubiquitinated membrane proteins and the Vps36 subunit of the downstream ESCRT II complex. The GLUE (GRAM-like, ubiquitin-binding on Eap45) domain extending beyond the core of the ESCRT II complex recognizes PtdIns3P-containing membranes, monoubiquitinated cargo and ESCRT I. The structure of this GLUE domain demonstrates that it has a split PH (pleckstrin homology) domain fold, with a non-typical phosphoinositide-binding pocket. Mutations in the lipid-binding pocket of the ESCRT II GLUE domain cause a strong defect in vacuolar protein sorting in yeast.     

Structural and mechanistic studies of VPS4 proteins

VPS4 ATPases function in multivesicular body formation and in HIV-1 budding. Here, we report the crystal structure of monomeric apo human VPS4B/SKD1 (hVPS4B), which is composed of five distinct elements: a poorly ordered N-terminal MIT domain that binds ESCRT-III substrates, large (mixed alpha/beta) and small (alpha) AAA ATPase domains that closely resemble analogous domains in the p97 D1 ATPase cassette, a three-stranded antiparallel beta domain inserted within the small ATPase domain, and a novel C-terminal helix. Apo hVPS4B and yeast Vps4p (yVps4p) proteins dimerized in solution, and assembled into larger complexes (10-12 subunits) upon ATP binding. Human and yeast adaptor proteins (LIP5 and yVta1p, respectively) bound the beta domains of the fully assembled hVPS4B and yVps4p proteins. We therefore propose that Vps4 proteins cycle between soluble, inactive low molecular weight complexes and active, membrane-associated double-ring structures that bind ATP and coassemble with LIP5/Vta1. Finally, HIV-1 budding was inhibited by mutations in a loop that projects into the center of the modeled hVPS4B rings, suggesting that hVPS4B may release the assembled ESCRT machinery by pulling ESCRT-III substrates up into the central pore.     

Cryo-EM structure of dodecameric Vps4p and its 2:1 complex with Vta1p

The type I AAA (ATPase associated with a variety of cellular activities) ATPase Vps4 and its co-factor Vta1p/LIP5 function in membrane remodeling events that accompany cytokinesis, multivesicular body biogenesis, and retrovirus budding, apparently by driving disassembly and recycling of membrane-associated ESCRT (endosomal sorting complex required for transport)-III complexes. Here, we present electron cryomicroscopy reconstructions of dodecameric yeast Vps4p complexes with and without their microtubule interacting and transport (MIT) N-terminal domains and Vta1p co-factors. The ATPase domains of Vps4p form a bowl-like structure composed of stacked hexameric rings. The two rings adopt dramatically different conformations, with the “upper” ring forming an open assembly that defines the sides of the bowl and the lower ring forming a closed assembly that forms the bottom of the bowl. The N-terminal MIT domains of the upper ring localize on the symmetry axis above the cavity of the bowl, and the binding of six extended Vta1p monomers causes additional density to appear both above and below the bowl. The structures suggest models in which Vps4p MIT and Vta1p domains engage ESCRT-III substrates above the bowl and help transfer them into the bowl to be pumped through the center of the dodecameric assembly.     

Vps4 regulates a subset of protein interactions at the multivesicular endosome

During endocytic transport, specific integral membrane proteins are sorted into intraluminal vesicles that bud from the limiting membrane of the endosome. This process, known as multivesicular body (MVB) sorting, is important for several important biological processes. Moreover, components of the MVB sorting machinery are implicated in virus budding. During MVB sorting, a cargo protein recruits components of the MVB sorting machinery from cytoplasmic pools and these sequentially assemble on the endosome. Disassembly of these proteins and recycling into the cytoplasm is critical for MVB sorting. Vacuolar protein sorting 4 (Vps4) is an AAA (ATPase associated with a variety of cellular activities) ATPase which has been proposed to play a critical role in disassembly of the MVB sorting machinery. However, the mechanism by which it disassembles the complex is not clear. Vps4 contains an N-terminal microtubule interacting and trafficking (MIT) domain, which has previously been shown to be required for recruitment to endosomes, and a single AAA ATPase domain, the activity of which is required for Vps4 function. In this study we have systematically characterized the interaction of Vps4 with other components of the MVB sorting machinery. We demonstrate that Vps4 interacts directly with Vps2 and Bro1. We also show that a subset of Vps4 interactions is regulated by ATP hydrolysis, and one interaction is regulated by ATP binding. Finally, we show that most proteins interact with the Vps4 MIT domain. Our studies indicate that the MIT domain has a dual role in substrate binding and recruitment to endosomes and indicate that Vps4 disassembles the MVB sorting machinery by direct effects on multiple proteins.     

Ist1 regulates Vps4 localization and assembly

The ESCRT protein complexes are recruited from the cytoplasm and assemble on the endosomal membrane into a protein network that functions in sorting of ubiquitinated transmembrane proteins into the multivesicular body (MVB) pathway. This transport pathway packages cargo proteins into vesicles that bud from the MVB limiting membrane into the lumen of the compartment and delivers these vesicles to the lysosome/vacuole for degradation. The dissociation of ESCRT machinery by the AAA-type ATPase Vps4 is a necessary late step in the formation of MVB vesicles. This ATP-consuming step is regulated by several Vps4-interacting proteins, including the newly identified regulator Ist1. Our data suggest that Ist1 has a dual role in the regulation of Vps4 activity: it localizes to the ESCRT machinery via Did2 where it positively regulates recruitment of Vps4 and it negatively regulates Vps4 by forming an Ist1-Vps4 heterodimer, in which Vps4 cannot bind to the ESCRT machinery. The activity of the MVB pathway might be in part determined by outcome of these two competing activities.     

A Common Substrate Recognition Mode Conserved between Katanin p60 and VPS4 Governs Microtubule Severing and Membrane Skeleton Reorganization

Katanin p60 (kp60), a microtubule-severing enzyme, plays a key role in cytoskeletal reorganization during various cellular events in an ATP-dependent manner. We show that a single domain isolated from the N terminus of mouse katanin p60 (kp60-NTD) binds to tubulin. The solution structure of kp60-NTD was determined by NMR. Although their sequence similarities were as low as 20%, the structure of kp60-NTD revealed a striking similarity to those of the microtubule interacting and trafficking (MIT) domains, which adopt anti-parallel three-stranded helix bundle. In particular, the arrangement of helices 2 and 3 is well conserved between kp60-NTD and the MIT domain from Vps4, which is a homologous protein that promotes disassembly of the endosomal sorting complexes required for transport III membrane skeleton complex. Mutation studies revealed that the positively charged surface formed by helices 2 and 3 binds tubulin. This binding mode resembles the interaction between the MIT domain of Vps4 and Vps2/CHMP1a, a component of endosomal sorting complexes required for transport III. Our results show that both the molecular architecture and the binding modes are conserved between two AAA-ATPases, kp60 and Vps4. A common mechanism is evolutionarily conserved between two distinct cellular events, one that drives microtubule severing and the other involving membrane skeletal reorganization.     

Split-ubiquitin two-hybrid assay to analyze protein-protein interactions at the endosome: application to Saccharomyces cerevisiae Bro1 interacting with ESCRT complexes, the Doa4 ubiquitin hydrolase, and the Rsp5 ubiquitin ligase

Targeting of membrane proteins into the lysosomal/vacuolar lumen for degradation requires their prior sorting into multivesicular bodies (MVB). The MVB sorting pathway depends on ESCRT-0, -I, -II, and -III protein complexes functioning on the endosomal membrane and on additional factors, such as Bro1/Alix and the ubiquitin ligase Rsp5/Nedd4. We used the split-ubiquitin two-hybrid assay to analyze the interaction partners of yeast Bro1 at its natural cellular location. We show that Bro1 interacts with ESCRT-I and -III components, including Vps23, the Saccharomyces cerevisiae homologue of human Tsg101. These interactions do not require the C-terminal proline-rich domain (PRD) of Bro1. Rather, this PRD interacts with the Doa4 deubiquitinating enzyme to recruit it to the endosome. This interaction is disrupted by a single amino acid substitution in the conserved ELC box motif in Doa4. The PRD of Bro1 also mediates an association with Rsp5, and this interaction appears to be conserved, as Alix, the human homologue of Bro1, coimmunoprecipitates with Nedd4 in yeast lysates. We further show that the Bro1 PRD domain is essential to MVB sorting of only cargo proteins whose sorting to the vacuolar lumen is dependent on their own ubiquitination and Doa4. The Bro1 region preceding the PRD, however, is required for MVB sorting of proteins irrespective of whether their targeting to the vacuole is dependent on their ubiquitination and Doa4. Our data indicate that Bro1 interacts with several ESCRT components and contributes via its PRD to associating ubiquitinating and deubiquitinating enzymes with the MVB sorting machinery.     

ESCRT-III family members stimulate Vps4 ATPase activity directly or via Vta1

The AAA-ATPase Vps4 is critical for function of the MVB sorting pathway, which in turn impacts cellular phenomena ranging from receptor downregulation to viral budding to cytokinesis. Vps4 dissociates ESCRTs from endosomal membranes during MVB sorting, but it is unclear how Vps4 ATPase activity is synchronized with ESCRT release. Vta1 potentiates Vps4 activity and interacts with ESCRT-III family members. We have investigated the impact of Vta1 and ESCRT-III family members on Vps4 ATPase activity. Two distinct mechanisms of Vps4 stimulation are described: Vps2 can directly stimulate Vps4 via its MIT domain, whereas Vps60 stimulates via Vta1. Moreover, Did2 can stimulate Vps4 by both mechanisms in distinct contexts. Recent structural determination of the ESCRT-III-binding region of Vta1 unexpectedly revealed a MIT-like region. These data support a model wherein a network of MIT and MIT-like domain interactions with ESCRT-III subunits contributes to the regulation of Vps4 activity during MVB sorting.     

Escrt-III: an endosome-associated heterooligomeric protein complex required for mvb sorting

The sorting of transmembrane proteins (e.g., cell surface receptors) into the multivesicular body (MVB) pathway to the lysosomal/vacuolar lumen requires the function of the ESCRT protein complexes. The soluble coiled-coil-containing proteins Vps2, Vps20, Vps24, and Snf7 are recruited from the cytoplasm to endosomal membranes where they oligomerize into a protein complex, ESCRT-III. ESCRT-III contains two functionally distinct subcomplexes. The Vps20-Snf7 subcomplex binds to the endosomal membrane, in part via the myristoyl group of Vps20. The Vps2-Vps24 subcomplex binds to the Vps20-Snf7 complex and thereby serves to recruit additional cofactors to this site of protein sorting. We provide evidence for a role for ESCRT-III in sorting and/or concentration of MVB cargoes.     

Pkh1/2-dependent phosphorylation of Vps27 regulates ESCRT-I recruitment to endosomes

Multivesicular endosomes (MVBs) are major sorting platforms for membrane proteins and participate in plasma membrane protein turnover, vacuolar/lysosomal hydrolase delivery, and surface receptor signal attenuation. MVBs undergo unconventional inward budding, which results in the formation of intraluminal vesicles (ILVs). MVB cargo sorting and ILV formation are achieved by the concerted function of endosomal sorting complex required for transport (ESCRT)-0 to ESCRT-III. The ESCRT-0 subunit Vps27 is a key player in this pathway since it recruits the other complexes to endosomes. Here we show that the Pkh1/Phk2 kinases, two yeast orthologues of the 3-phosphoinositide–dependent kinase, phosphorylate directly Vps27 in vivo and in vitro. We identify the phosphorylation site as the serine 613 and demonstrate that this phosphorylation is required for proper Vps27 function. Indeed, in pkh-ts temperature-sensitive mutant cells and in cells expressing vps27^S613A, MVB sorting of the carboxypeptidase Cps1 and of the α-factor receptor Ste2 is affected and the Vps28–green fluorescent protein ESCRT-I subunit is mainly cytoplasmic. We propose that Vps27 phosphorylation by Pkh1/2 kinases regulates the coordinated cascade of ESCRT complex recruitment at the endosomal membrane.     

1H, 13C and 15N resonance assignments of the N-terminal domain of Vta1–Vps60 peptide complex

Vta1 and Vps60 are two ESCRT associated proteins, their direct interaction enhances Vps4 ATPase activity. The N-terminal domain of Vta1 (residues 1–167aa, named as Vta1NTD) contains two tandem MIT domains, which specifically recognize Vps60 and Did2 but not other ESCRT-III subunits. The fragment Vps60 (128–186aa) was reported to display full activity of Vps60, which stimulates Vps4 ATPase in a Vta1-dependent manner. To study the structural basis for the interaction between Vta1 and Vps60, as a first step, here, we report the resonance assignments of the sequential backbone atoms and the side chains of the residues in the two components of Vta1NTD/Vps60_128–186 complex at pH 7.0 and 20 °C (BMRB No. 18521).     

A systematic analysis of human CHMP protein interactions: additional MIT domain-containing proteins bind to multiple components of the human ESCRT III complex

In Saccharomyces cerevisiae 6 closely related proteins (Did2p, Vps2p, Vps24p, Vps32p, Vps60p, Vps20p) form part of the extended ESCRT III complex. This complex is required for the formation of multivesicular bodies and the degradation of internalized transmembrane receptor proteins. In contrast the human genome encodes 10 homologous proteins (CHMP1A (approved gene symbol PCOLN3), 1B, 2A, 2B, 3 (approved gene symbol VPS24), 4A, 4B, 4C, 5, and 6). In this study we have performed a series of protein interaction experiments to generate a more comprehensive picture of the human CHMP protein-interaction network. Our results describe novel interactions between known components of the human ESCRT III complex and identify a range of putative binding partners, which may indicate new ways in which the function of human CHMP proteins may be regulated. In particular, we show that two further MIT domain-containing proteins (AMSH/STAMBP and LOC129531) interact with multiple components of the human ESCRT III complex.     

Ubiquitin binding by the CUE domain promotes endosomal localization of the Rab5 GEF Vps9

Vps9 and Muk1 are guanine nucleotide exchange factors (GEFs) in Saccharomyces cerevisiae that regulate membrane trafficking in the endolysosomal pathway by activating Rab5 GTPases. We show that Vps9 is the primary Rab5 GEF required for biogenesis of late endosomal multivesicular bodies (MVBs). However, only Vps9 (but not Muk1) is required for the formation of aberrant class E compartments that arise upon dysfunction of endosomal sorting complexes required for transport (ESCRTs). ESCRT dysfunction causes ubiquitinated transmembrane proteins to accumulate at endosomes, and we demonstrate that endosomal recruitment of Vps9 is promoted by its ubiquitin-binding CUE domain. Muk1 lacks ubiquitin-binding motifs, but its fusion to the Vps9 CUE domain allows Muk1 to rescue endosome morphology, cargo trafficking, and cellular stress-tolerance phenotypes that result from loss of Vps9 function. These results indicate that ubiquitin binding by the CUE domain promotes Vps9 function in endolysosomal membrane trafficking via promotion of localization.     

Interactions of ubiquitin and CHMP5 with the V domain of HD-PTP reveals role for regulation of Vps4 ATPase

The family of Bro1 proteins coordinates the activity of the Endosomal Sorting Complexes Required for Transport (ESCRTs) to mediate a number of membrane remodeling events. These events culminate in membrane scission catalyzed by ESCRT-III, whose polymerization and disassembly is controlled by the AAA-ATPase, Vps4. Bro1-family members Alix and HD-PTP as well as yeast Bro1 have central “V” domains that noncovalently bind Ub and connect ubiquitinated proteins to ESCRT-driven functions such as the incorporation of ubiquitinated membrane proteins into intralumenal vesicles of multivesicular bodies. Recently, it was discovered that the V domain of yeast Bro1 binds the MIT domain of Vps4 to stimulate its ATPase activity. Here we determine the structural basis for how the V domain of human HD-PTP binds ubiquitin. The HD-PTP V domain also binds the MIT domain of Vps4, and ubiquitin binding to the HD-PTP V domain enhances its ability to stimulate Vps4 ATPase activity. Additionally, we found that V domains of both HD-PTP and Bro1 bind CHMP5 and Vps60, respectively, providing another potential molecular mechanism to alter Vps4 activity. These data support a model whereby contacts between ubiquitin, ESCRT-III, and Vps4 by V domains of the Bro1 family may coordinate late events in ESCRT-driven membrane remodeling events.     

Noncanonical Role for the Host Vps4 AAA+ ATPase ESCRT Protein in the Formation of Tomato Bushy Stunt Virus Replicase

Assembling of the membrane-bound viral replicase complexes (VRCs) consisting of viral- and host-encoded proteins is a key step during the replication of positive-stranded RNA viruses in the infected cells. Previous genome-wide screens with Tomato bushy stunt tombusvirus (TBSV) in a yeast model host have revealed the involvement of eleven cellular ESCRT (endosomal sorting complexes required for transport) proteins in viral replication. The ESCRT proteins are involved in endosomal sorting of cellular membrane proteins by forming multiprotein complexes, deforming membranes away from the cytosol and, ultimately, pinching off vesicles into the lumen of the endosomes. In this paper, we show an unexpected key role for the conserved Vps4p AAA+ ATPase, whose canonical function is to disassemble the ESCRT complexes and recycle them from the membranes back to the cytosol. We find that the tombusvirus p33 replication protein interacts with Vps4p and three ESCRT-III proteins. Interestingly, Vps4p is recruited to become a permanent component of the VRCs as shown by co-purification assays and immuno-EM. Vps4p is co-localized with the viral dsRNA and contacts the viral (+)RNA in the intracellular membrane. Deletion of Vps4p in yeast leads to the formation of crescent-like membrane structures instead of the characteristic spherule and vesicle-like structures. The in vitro assembled tombusvirus replicase based on cell-free extracts (CFE) from vps4Δ yeast is highly nuclease sensitive, in contrast with the nuclease insensitive replicase in wt CFE. These data suggest that the role of Vps4p and the ESCRT machinery is to aid building the membrane-bound VRCs, which become nuclease-insensitive to avoid the recognition by the host antiviral surveillance system and the destruction of the viral RNA. Other (+)RNA viruses of plants and animals might also subvert Vps4p and the ESCRT machinery for formation of VRCs, which require membrane deformation and spherule formation.     

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.