Yeast dynamin Vps1 and amphiphysin Rvs167 function together during endocytosis

Dynamins are a conserved family of proteins involved in many membrane fusion and fission events. Previously, the dynamin-related protein Vps1 was shown to localize to endocytic sites, and yeast carrying deletions for genes encoding both the BAR domain protein Rvs167 and Vps1 had a more severe endocytic scission defect than either deletion alone. Vps1 and Rvs167 localize to endocytic sites at the onset of invagination and disassemble concomitant with inward vesicle movement. Rvs167-GFP localization is reduced in cells lacking vps1 suggesting that Vps1 influences Rvs167 association with the endocytic complex. Unlike classical dynamins, Vps1 does not have a proline-arginine domain that could interact with SH3 domain-containing proteins. Thus, while Rvs167 has an SH3 domain, it is not clear how an interaction would be mediated. Here, we demonstrate an interaction between Rvs167 SH3 domain and the single type I SH3-binding motif in Vps1. Mutant Vps1 that cannot bind Rvs167 rescues all membrane fusion/fission functions associated with Vps1 except for endocytic function, demonstrating the specificity and mechanistic importance of the interaction. In vitro, an Rvs161/Rvs167 heterodimer can disassemble Vps1 oligomers. Overall, the data support the idea that Vps1 and the amphiphysins function together to mediate scission during endocytosis in yeast.     

Correlated fluorescence and 3D electron microscopy with high sensitivity and spatial precision

Correlative electron and fluorescence microscopy has the potential to elucidate the ultrastructural details of dynamic and rare cellular events, but has been limited by low precision and sensitivity. Here we present a method for direct mapping of signals originating from ～20 fluorescent protein molecules to 3D electron tomograms with a precision of less than 100 nm. We demonstrate that this method can be used to identify individual HIV particles bound to mammalian cell surfaces. We also apply the method to image microtubule end structures bound to mal3p in fission yeast, and demonstrate that growing microtubule plus-ends are flared in vivo. We localize Rvs167 to endocytic sites in budding yeast, and show that scission takes place halfway through a 10-s time period during which amphiphysins are bound to the vesicle neck. This new technique opens the door for direct correlation of fluorescence and electron microscopy to visualize cellular processes at the ultrastructural scale.     

Identification and Characterization of Rvs162/Rvs167-3, a Novel N-BAR Heterodimer in the Human Fungal Pathogen Candida albicans

Membrane reshaping resides at the core of many important cellular processes, and among its mediators are the BAR (Bin, Amphiphysin, Rvs) domain-containing proteins. We have explored the diversity and function of the Rvs BAR proteins in Candida albicans and identified a novel family member, Rvs167-3 (orf19.1861). We show that Rvs167-3 specifically interacts with Rvs162 to form a stable BAR heterodimer able to bind liposomes in vitro. A second, distinct heterodimer is formed by the canonical BAR proteins Rvs161 and Rvs167. Purified Rvs161/Rvs167 complex also binds liposomes, indicating that C. albicans expresses two functional BAR heterodimers. We used live-cell imaging to localize green fluorescent protein (GFP)-tagged Rvs167-3 and Rvs167 and show that both proteins concentrate in small cortical spots. However, while Rvs167 strictly colocalizes with the endocytic marker protein Abp1, we do not observe any colocalization of Rvs167-3 with sites of endocytosis marked by Abp1. Furthermore, the rvs167-3Δ/Δ mutant is not defective in endocytosis and strains lacking Rvs167-3 or its partner Rvs162 do not display increased sensitivity to high salt concentrations or decreased cell wall integrity, phenotypes which have been observed for rvs167Δ/Δ and rvs161Δ/Δ strains and which are linked to endocytosis defects. Taken together, our results indicate different roles for the two BAR heterodimers in C. albicans: the canonical Rvs161/Rvs167 heterodimer functions in endocytosis, whereas the novel Rvs162/Rvs167-3 heterodimer seems not to be involved in this process. Nevertheless, despite their different roles, our phenotypic analysis revealed a genetic interaction between the two BAR heterodimers, suggesting that they may have related but distinct membrane-associated functions.     

Hof1 and Rvs167 Have Redundant Roles in Actomyosin Ring Function during Cytokinesis in Budding Yeast

The Hof1 protein (Homologue of Fifteen) regulates formation of the primary septum during cytokinesis in the budding yeast Saccharomyces cerevisiae, whereas the orthologous Cdc15 protein in fission yeast regulates the actomyosin ring by using its F-BAR domain to recruit actin nucleators to the cleavage site. Here we show that budding yeast Hof1 also contributes to actin ring assembly in parallel with the Rvs167 protein. Simultaneous deletion of the HOF1 and RVS167 genes is lethal, and cells fail to assemble the actomyosin ring as they progress through mitosis. Although Hof1 and Rvs167 are not orthologues, they both share an analogous structure, with an F-BAR or BAR domain at the amino terminus, capable of inducing membrane curvature, and SH3 domains at the carboxyl terminus that bind to specific proline-rich targets. The SH3 domain of Rvs167 becomes essential for assembly of the actomyosin ring in cells lacking Hof1, suggesting that it helps to recruit a regulator of the actin cytoskeleton. This new function of Rvs167 appears to be independent of its known role as a regulator of the Arp2/3 actin nucleator, as actin ring assembly is not abolished by the simultaneous inactivation of Hof1 and Arp2/3. Instead we find that recruitment to the bud-neck of the Iqg1 actin regulator is defective in cells lacking Hof1 and Rvs167, though future studies will be needed to determine if this reflects a direct interaction between these factors. The redundant role of Hof1 in actin ring assembly suggests that the mechanism of actin ring assembly has been conserved to a greater extent across evolution than anticipated previously.     

A Complex Network of Interactions between Mitotic Kinases,Phosphatases and ESCRT Proteins Regulates Septation and Membrane Trafficking in S. pombe

Cytokinesis and cell separation are critical events in the cell cycle. We show that Endosomal Sorting Complex Required for Transport (ESCRT) genes are required for cell separation in Schizosaccharomyces pombe. We identify genetic interactions between ESCRT proteins and polo and aurora kinases and Cdc14 phosphatase that manifest as impaired growth and exacerbated defects in septation, suggesting that the encoded proteins function together to control these processes. Furthermore, we observed defective endosomal sorting in mutants of plo1, ark1 and clp1, as has been reported for ESCRT mutants, consistent with a role for these kinases in the control of ESCRT function in membrane traffic. Multiple observations indicate functional interplay between polo and ESCRT components: firstly, two-hybrid in vivo interactions are reported between Plo1p and Sst4p, Vps28p, Vps25p, Vps20p and Vps32p; secondly, co-immunoprecipitation of human homologues of Vps20p, Vps32p, Vps24p and Vps2p by human Plk1; and thirdly, in vitro phosphorylation of budding yeast Vps32p and Vps20p by polo kinase. Two-hybrid analyses also identified interactions between Ark1p and Vps20p and Vps32p, and Clp1p and Vps28p. These experiments indicate a network of interactions between ESCRT proteins, plo1, ark1 and clp1 that coordinate membrane trafficking and cell separation in fission yeast.     

Dissecting BAR Domain Function in the Yeast Amphiphysins Rvs161 and Rvs167 during Endocytosis

BAR domains are protein modules that bind to membranes and promote membrane curvature. One type of BAR domain, the N-BAR domain, contains an additional N-terminal amphipathic helix, which contributes to membrane-binding and bending activities. The only known N-BAR-domain proteins in the budding yeast Saccharomyces cerevisiae, Rvs161 and Rvs167, are required for endocytosis. We have explored the mechanism of N-BAR-domain function in the endocytosis process using a combined biochemical and genetic approach. We show that the purified Rvs161–Rvs167 complex binds to liposomes in a curvature-independent manner and promotes tubule formation in vitro. Consistent with the known role of BAR domain polymerization in membrane bending, we found that Rvs167 BAR domains interact with each other at cortical actin patches in vivo. To characterize N-BAR-domain function in endocytosis, we constructed yeast strains harboring changes in conserved residues in the Rvs161 and Rvs167 N-BAR domains. In vivo analysis of the rvs endocytosis mutants suggests that Rvs proteins are initially recruited to sites of endocytosis through their membrane-binding ability. We show that inappropriate regulation of complex sphingolipid and phosphoinositide levels in the membrane can impinge on Rvs function, highlighting the relationship between membrane components and N-BAR-domain proteins in vivo.     

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.     

The dynamin-related protein Vps1 regulates vacuole fission,fusion and tubulation in the fission yeast,Schizosaccharomyces pombe

Fission yeast cells lacking the dynamin-related protein (DRP) Vps1 had smaller vacuoles with reduced capacity for both fusion and fission in response to hypotonic and hypertonic conditions respectively. vps1Δ cells showed normal vacuolar protein sorting, actin organisation and endocytosis. Over-expression of vps1 transformed vacuoles from spherical to tubular. Tubule formation was enhanced in fission conditions and required the Rab protein Ypt7. Vacuole tubulation by Vps1 was more extensive in the absence of a second DRP, Dnm1. Both dnm1Δ and the double mutant vps1Δ dnm1Δ showed vacuole fission defects similar to that of vps1Δ. Over-expression of vps1 in dnm1Δ, or of dnm1 in vps1Δ failed to rescue this phenotype. Over-expression of dnm1 in wild-type cells, on the other hand, induced vacuole fission. Our results are consistent with a model of vacuole fission in which Vps1 creates a tubule of an appropriate diameter for subsequent scission by Dnm1.     

Characterization of the yeast amphiphysins Rvs161p and Rvs167p reveals roles for the Rvs heterodimer in vivo

We have used comprehensive synthetic lethal screens and biochemical assays to examine the biological role of the yeast amphiphysin homologues Rvs161p and Rvs167p, two proteins that play a role in regulation of the actin cytoskeleton, endocytosis, and sporulation. We found that unlike some forms of amphiphysin, Rvs161p-Rvs167p acts as an obligate heterodimer during vegetative growth and neither Rvs161p nor Rvs167p forms a homodimer in vivo. RVS161 and RVS167 have an identical set of 49 synthetic lethal interactions, revealing functions for the Rvs proteins in cell polarity, cell wall synthesis, and vesicle trafficking as well as a shared role in mating. Consistent with these roles, we show that the Rvs167p-Rvs161p heterodimer, like its amphiphysin homologues, can bind to phospholipid membranes in vitro, suggesting a role in vesicle formation and/or fusion. Our genetic screens also reveal that the interaction between Abp1p and the Rvs167p Src homology 3 (SH3) domain may be important under certain conditions, providing the first genetic evidence for a role for the SH3 domain of Rvs167p. Our studies implicate heterodimerization of amphiphysin family proteins in various functions related to cell polarity, cell integrity, and vesicle trafficking during vegetative growth and the mating response.     

Distinct complexes of yeast Snx4 family SNX‐BARs mediate retrograde trafficking of Snc1 and Atg27

The yeast SNX4 sub‐family of sorting nexin containing a Bin‐Amphiphysin‐Rvs domain (SNX‐BAR) proteins, Snx4/Atg24, Snx41 and Atg20/Snx42, are required for endocytic recycling and selective autophagy. Here, we show that Snx4 forms 2 functionally distinct heterodimers: Snx4‐Atg20 and Snx4‐Snx41. Each heterodimer coats an endosome‐derived tubule that mediates retrograde sorting of distinct cargo; the v‐SNARE, Snc1, is a cargo of the Snx4‐Atg20 pathway, and Snx4‐Snx41 mediates retrograde sorting of Atg27, an integral membrane protein implicated in selective autophagy. Live cell imaging of individual endosomes shows that Snx4 and the Vps5‐Vps17 retromer SNX‐BAR heterodimer operate concurrently on a maturing endosome. Consistent with this, the yeast dynamin family protein, Vps1, which was previously shown to promote fission of retromer‐coated tubules, promotes fission of Snx4‐Atg20 coated tubules. The results indicate that the yeast SNX‐BAR proteins coat 3 distinct types of endosome‐derived carriers that mediate endosome‐to‐Golgi retrograde trafficking.      

Inactivation of Tor proteins affects the dynamics of endocytic proteins in early stage of endocytosis

Tor2 is an activator of the Rom2/Rho1 pathway that regulates α-factor internalization. Since the recruitment of endocytic proteins such as actin-binding proteins and the amphiphysins precedes the internalization of α-factor, we hypothesized that loss of Tor function leads to an alteration in the dynamics of the endocytic proteins. We report here that endocytic proteins, Abp1 and Rvs167, are less recruited to endocytic sites not only in tor2 but also tor1 mutants. Furthermore, we found that the endocytic proteins Rvs167 and Sjl2 are completely mistargeted to the cytoplasm in tor1Δtor2 ^ts double mutant cells. We also demonstrate here that the efficiency of endocytic internalization or scission in all tor mutants was drastically decreased. In agreement with the Sjl2 mislocalization, we found that in tor1Δtor2 ^ts double mutant cells, as well as other tor mutant cells, the overall PIP₂ level was dramatically increased. Finally, the cell wall chitin content in tor2 ^ts and tor1Δtor2ts mutant cells was also significantly increased. Taken together, both functional Tor proteins, Tor1 and Tor2, are essentially required for proper endocytic protein dynamics at the early stage of endocytosis.     

Cooperative Recruitment of Dynamin and BIN/Amphiphysin/Rvs (BAR) Domain-containing Proteins Leads to GTP-dependent Membrane Scission

Dynamin mediates various membrane fission events, including the scission of clathrin-coated vesicles. Here, we provide direct evidence for cooperative membrane recruitment of dynamin with the BIN/amphiphysin/Rvs (BAR) proteins, endophilin and amphiphysin. Surprisingly, endophilin and amphiphysin recruitment to membranes was also dependent on binding to dynamin due to auto-inhibition of BAR-membrane interactions. Consistent with reciprocal recruitment in vitro, dynamin recruitment to the plasma membrane in cells was strongly reduced by concomitant depletion of endophilin and amphiphysin, and conversely, depletion of dynamin dramatically reduced the recruitment of endophilin. In addition, amphiphysin depletion was observed to severely inhibit clathrin-mediated endocytosis. Furthermore, GTP-dependent membrane scission by dynamin was dramatically elevated by BAR domain proteins. Thus, BAR domain proteins and dynamin act in synergy in membrane recruitment and GTP-dependent vesicle scission.     

GTP-stimulated membrane fission by the N-BAR protein AMPH-1

Membrane-enclosed transport carriers sort biological molecules between stations in the cell in a dynamic process that is fundamental to the physiology of eukaryotic organisms. While much is known about the formation and release of carriers from specific intracellular membranes, the mechanism of carrier formation from the recycling endosome, a compartment central to cellular signaling, remains to be resolved. In Caenorhabditis elegans, formation of transport carriers from the recycling endosome requires the dynamin-like, Eps15-homology domain (EHD) protein, RME-1, functioning with the Bin/Amphiphysin/Rvs (N-BAR) domain protein, AMPH-1. Here we show, using a free-solution single-particle technique known as burst analysis spectroscopy (BAS), that AMPH-1 alone creates small, tubular-vesicular products from large, unilamellar vesicles by membrane fission. Membrane fission requires the amphipathic H0 helix of AMPH-1 and is slowed in the presence of RME-1. Unexpectedly, AMPH-1-induced membrane fission is stimulated in the presence of GTP. Furthermore, the GTP-stimulated membrane fission activity seen for AMPH-1 is recapitulated by the heterodimeric N-BAR amphiphysin protein from yeast, Rvs161/167p, strongly suggesting that GTP-stimulated membrane fission is a general property of this important class of N-BAR proteins.     

The Oligomeric State of the Active Vps4 AAA ATPase

The cellular ESCRT (endosomal sorting complexes required for transport) pathway drives membrane constriction toward the cytosol and effects membrane fission during cytokinesis, endosomal sorting, and the release of many enveloped viruses, including the human immunodeficiency virus. A component of this pathway, the AAA ATPase Vps4, provides energy for pathway progression. Although it is established that Vps4 functions as an oligomer, subunit stoichiometry and other fundamental features of the functional enzyme are unclear. Here, we report that although some mutant Vps4 proteins form dodecameric assemblies, active wild-type Saccharomyces cerevisiae and Sulfolobus solfataricus Vps4 enzymes can form hexamers in the presence of ATP and ADP, as assayed by size-exclusion chromatography and equilibrium analytical ultracentrifugation. The Vta1p activator binds hexameric yeast Vps4p without changing the oligomeric state of Vps4p, implying that the active Vta1p–Vps4p complex also contains a single hexameric ring. Additionally, we report crystal structures of two different archaeal Vps4 homologs, whose structures and lattice interactions suggest a conserved mode of oligomerization. Disruption of the proposed hexamerization interface by mutagenesis abolished the ATPase activity of archaeal Vps4 proteins and blocked Vps4p function in S. cerevisiae. These data challenge the prevailing model that active Vps4 is a double-ring dodecamer, and argue that, like other type I AAA ATPases, Vps4 functions as a single ring with six subunits.     

Characterizing the sphingolipid signaling pathway that remediates defects associated with loss of the yeast amphiphysin-like orthologs, Rvs161p and Rvs167p

Loss of function of either the RVS161 or RVS167 Saccharomyces cerevisiae amphiphysin-like gene confers similar growth phenotypes that can be suppressed by mutations in sphingolipid biosynthesis. We performed a yeast two-hybrid screen using Rvs161p as bait to uncover proteins involved in this sphingolipid-dependent suppressor pathway. In the process, we have demonstrated a direct physical interaction between Rvs167p and the two-hybrid interacting proteins, Acf2p, Gdh3p, and Ybr108wp, while also elucidating the Rvs167p amino acid domains to which these proteins bind. By using subcellular fractionation, we demonstrate that Rvs167p, Ybr108wp, Gdh3p, and Acf2p all localize to Rvs161p-containing lipid rafts, thus placing them within a single compartment that should facilitate their interactions. Moreover, our results suggest that Acf2p and Gdh3p functions are needed for suppressor pathway activity. To determine pathway mechanisms further, we examined the localization of Rvs167p in suppressor mutants. These studies reveal roles for Rvs161p and the very long chain fatty acid elongase, Sur4p, in the localization and/or stability of Rvs167p. Previous yeast studies showed that rvs defects could be suppressed by changes in sphingolipid metabolism brought about by deleting SUR4 (Desfarges, L., Durrens, P., Juguelin, H., Cassagne, C., Bonneu, M., and Aigle, M. (1993) Yeast 9, 267-277). Using rvs167 sur4 and rvs161 sur4 double null cells as models to study suppressor pathway activity, we demonstrate that loss of SUR4 does not remediate the steady-state actin cytoskeletal defects of rvs167 or rvs161 cells. Moreover, suppressor activity does not require the function of the actin-binding protein, Abp1p, or Sla1p, a protein that is thought to regulate assembly of the cortical actin cytoskeleton. Based on our results, we suggest that sphingolipid-dependent suppression of rvs defects may not work entirely through regulating changes in actin organization.     

In vivo analysis of the domains of yeast Rvs167p suggests Rvs167p function is mediated through multiple protein interactions.

Morphological changes during cell division in the yeast Saccharomyces cerevisiae are controlled by cell-cycle regulators. The Pcl-Pho85p kinase complex has been implicated in the regulation of the actin cytoskeleton at least in part through Rvs167p. Rvs167p consists of three domains called BAR, GPA, and SH3. Using a two-hybrid assay, we demonstrated that each region of Rvs167p participates in protein-protein interactions: the BAR domain bound the BAR domain of another Rvs167p protein and that of Rvs161p, the GPA region bound Pcl2p, and the SH3 domain bound Abp1p. We identified Rvs167p as a Las17p/Bee1p-interacting protein in a two-hybrid screen and showed that Las17p/Bee1p bound the SH3 domain of Rvs167p. We tested the extent to which the Rvs167p protein domains rescued phenotypes associated with deletion of RVS167: salt sensitivity, random budding, and endocytosis and sporulation defects. The BAR domain was sufficient for full or partial rescue of all rvs167 mutant phenotypes tested but not required for the sporulation defect for which the SH3 domain was also sufficient. Overexpression of Rvs167p inhibits cell growth. The BAR domain was essential for this inhibition and the SH3 domain had only a minor effect. Rvs167p may link the cell cycle regulator Pcl-Pho85p kinase and the actin cytoskeleton. We propose that Rvs167p is activated by phosphorylation in its GPA region by the Pcl-Pho85p kinase. Upon activation, Rvs167p enters a multiprotein complex, making critical contacts in its BAR domain and redundant or minor contacts with its SH3 domain.     

Regulation of the yeast amphiphysin homologue Rvs167p by phosphorylation

The yeast amphiphysin homologue Rvs167p plays a role in regulation of the actin cytoskeleton, endocytosis, and sporulation. Rvs167p is a phosphoprotein in vegetatively growing cells and shows increased phosphorylation upon treatment with mating pheromone. Previous work has shown that Rvs167p can be phosphorylated in vitro by the cyclin-dependent kinase Pho85p complexed with its cyclin Pcl2p. Using chymotryptic phosphopeptide mapping, we have identified the sites on which Rvs167p is phosphorylated in vitro by Pcl2p-Pho85p. We have shown that these same sites are phosphorylated in vivo during vegetative growth and that phosphorylation at two of these sites is Pcl-Pho85p dependent. In cells treated with mating pheromone, the MAP kinase Fus3p is needed for full phosphorylation of Rvs167p. Functional genomics and genetics experiments revealed that mutation of other actin cytoskeleton genes compromises growth of a strain in which phosphorylation of Rvs167p is blocked by mutation. Phosphorylation of Rvs167p inhibits its interaction in vitro with Las17p, an activator of the Arp2/3 complex, as well as with a novel protein, Ymr192p. Our results suggest that phosphorylation of Rvs167p by a cyclin-dependent kinase and by a MAP kinase is an important mechanism for regulating protein complexes involved in actin cytoskeleton function.     

A Dynamin Homolog Promotes the Transition from Hemifusion to Content Mixing in Intracellular Membrane Fusion

The convergence of the antagonistic reactions of membrane fusion and fission at the hemifusion/hemifission intermediate has generated a captivating enigma of whether Soluble N‐ethylmaleimide sensitive factor Attachment Protein Receptor (SNAREs) and dynamin have unusual counter‐functions in fission and fusion, respectively. SNARE‐mediated fusion and dynamin‐driven fission are fundamental membrane flux reactions known to occur during ubiquitous cellular communication events such as exocytosis, endocytosis and vesicle transport. Here we demonstrate the influence of the dynamin homolog Vps1 (Vacuolar protein sorting 1) on lipid mixing and content mixing properties of yeast vacuoles, and on the incorporation of SNAREs into fusogenic complexes. We propose a novel concept that Vps1, through its oligomerization and SNARE domain binding, promotes the hemifusion‐content mixing transition in yeast vacuole fusion by increasing the number of trans‐SNAREs .      

Association of the endosomal sorting complex ESCRT-II with the Vps20 subunit of ESCRT-III generates a curvature-sensitive complex capable of nucleating ESCRT-III filaments

The scission of membranes necessary for vesicle biogenesis and cytokinesis is mediated by cytoplasmic proteins, which include members of the ESCRT (endosomal sorting complex required for transport) machinery. During the formation of intralumenal vesicles that bud into multivesicular endosomes, the ESCRT-II complex initiates polymerization of ESCRT-III subunits essential for membrane fission. However, mechanisms underlying the spatial and temporal regulation of this process remain unclear. Here, we show that purified ESCRT-II binds to the ESCRT-III subunit Vps20 on chemically defined membranes in a curvature-dependent manner. Using a combination of liposome co-flotation assays, fluorescence-based liposome interaction studies, and high-resolution atomic force microscopy, we found that the interaction between ESCRT-II and Vps20 decreases the affinity of ESCRT-II for flat lipid bilayers. We additionally demonstrate that ESCRT-II and Vps20 nucleate flexible filaments of Vps32 that polymerize specifically along highly curved membranes as a single string of monomers. Strikingly, Vps32 filaments are shown to modulate membrane dynamics in vitro, a prerequisite for membrane scission events in cells. We propose that a curvature-dependent assembly pathway provides the spatial regulation of ESCRT-III to fuse juxtaposed bilayers of elevated curvature.     

A membrane-associated complex containing the Vps15 protein kinase and the Vps34 PI 3-kinase is essential for protein sorting to the yeast lysosome-like vacuole.

The Vps15 protein kinase and the Vps34 phosphatidylinositol 3-kinase (PI 3-kinase) are required for the sorting of soluble hydrolases to the yeast vacuole. Over-production of Vps34p suppresses the growth and vacuolar protein sorting defects associated with vps15 kinase domain mutants, suggesting that Vps15p and Vps34p functionally interact. Subcellular fractionation and sucrose density gradients indicate that Vps15p is responsible for the association of Vps34p with an intracellular membrane fraction. Chemical cross-linking and native immunoprecipitation experiments demonstrate that Vps15p and Vps34p interact as components of a hetero-oligomeric protein complex. In addition, we show that an intact Vps15 protein kinase domain is required for activation of the Vps34 PI 3-kinase, suggesting that the Vps34 lipid kinase is regulated by a Vps15p-mediated protein phosphorylation event. We propose that Vps15p and Vps34p function together as components of a membrane-associated signal transduction complex that regulates intracellular protein trafficking decisions through protein and lipid phosphorylation events.