Acinus: A nuclear regulator of autophagy and endocytic trafficking

Fusion with lysosomes is the common last step of endocytic trafficking and autophagy. Accordingly, several proteins are required in both pathways for cargoes to reach their destinations. Among these proteins, Drosophila Acinus stands out, as it exerts opposite effects on these two pathways, and thus establishes a new paradigm. Loss of Acinus function destabilizes early endosomes, thereby promoting the delivery of their cargo to lysosomes. By contrast, the maturation of autophagosomes to autolysosomes is inhibited in acn mutant cells. The increase in autophagy upon Acinus overexpression and its location to the nucleus are consistent with Acinus being a novel regulator of autophagy.Key words: fat body, endosomes, lysosomes, nuclear protein, Notch signaling, EGF ReceptorMuch of the core machinery that is required for the formation and maturation of autophagosomes and endosomes has been identified by genetic screens in yeast. But as both types of organelles are charged with more complex functions in multicellular organisms, it is not surprising to find additional layers of regulation imposed on them. One such regulatory element was revealed by a genetic screen we conducted in Drosophila.The screen''s original idea was to take advantage of the observation that many proteins acting in trafficking to lysosomes also function in the biogenesis of lysosome-related organelles. Among these, the pigment granules—responsible for the characteristic color of the fly eye—are easily scored for defects. Thus, we set up a primary screen for eye color mutants. Among the more than 500 original hits, a secondary screen identified those mutants that altered endocytic trafficking. Importantly, the genetic tool kit assembled by the fly community allowed us to screen homozygous mutant eyes in otherwise heterozygous flies. This schema made it possible to identify mutations that are homozygous lethal as one might expect for null alleles of genes required for lysosomal delivery.One of the unexpected genes identified by this screen was acinus (acn). The Acn protein lacks any domain signatures and is most similar to human Acinus, which had been implicated in the destruction of chromatin during apoptosis. It is not clear yet whether the Drosophila protein contributes to this function as well, but in acn null alleles chromatin condensation and fragmentation during apoptosis appear normal.There is, however, a profound effect on endocytic trafficking, as acn is required for stabilization of early endosomes. Staining for endocytosed ligands, such as Boss or Delta, is drastically reduced, concomitant with a reduction in early endosomes marked by Rab5 or the SNARE Avl. By contrast, late endosomes marked by Rab7 appear normal. These changes do not represent a block in the initial internalization of the ligands, as inhibition of lysosomal degradation reveals the same accumulation of internalized ligands in wild-type and acn mutant cells.Reduced stability of early endosomes also causes reduced signaling from EGF receptors and Notch, consistent with the emerging notion that signaling from these receptors may be linked to their uptake into early endosomes.Many mutants that disrupt endocytic trafficking also affect autophagy. We found that this theme extends to acn. The most accessible form of autophagy in Drosophila is found in fat bodies after a short period of starvation. Activation of the AKT1/TOR pathway triggers the formation of autophagosomes, which mature into autolysosomes by fusing with lysosomes. Loss of acn interferes with this maturation step, as shown by the reduction in LysoTracker staining and also by quantitative electron microscopy. Consistent with an effect on the maturation of autophagosomes, acn is required downstream of TOR signaling. For example, expression of dominant-negative TOR kinase is a powerful tool to induce autophagy in the fat body of wild-type, but not acn larvae.Interestingly, overexpression of Acn induces autophagy. This does not appear to be merely a side effect. Ubiquitous expression of Acn is lethal, but flies survive when autophagy is suppressed by knockdown of ATG5, a core element of the autophagy machinery. We find that this enhanced autophagy is also independent of the TOR pathway.Taken together, this analysis of the first null mutant of an acinus gene in any system reveals its function as a regulator of endosomal and autophagosomal dynamics, modulating developmental signaling and the cellular response to starvation. Our investigation of acn loss-of-function phenotypes reveals defects in membrane trafficking during endocytosis and autophagy. We were therefore surprised that Acn protein localized to the nucleus, and that we failed to detect any consistent localization to endocytic or autophagic structures. This unexpected finding was further tested with transgenes expressing Myc-tagged Acn in the context of a genomic rescue construct. This tagged protein, under control of its endogenous enhancer/promoter elements, rescued all aspects of Acn function, and, nevertheless, localized to the nucleus, rather than any endosomal compartment.These findings suggest that the mechanism by which Acinus proteins modify endocytosis and autophagy may be indirect. One model for such an indirect effect is suggested by the interaction of mammalian Acinus proteins with several RNA binding proteins. Modulation of the levels or structure of RNAs that encode specific elements of the endocytosis or autophagy pathways may constitute an exciting new element of their regulation. Testing this possibility and identifying potential targets regulated by this Acn-dependent mechanism are important challenges that we have just begun to address.     

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.     

The Atg6/Vps30/Beclin 1 ortholog BEC-1 mediates endocytic retrograde transport in addition to autophagy in C. elegans

Autophagy and endocytosis are dynamic and tightly regulated processes that contribute to many fundamental aspects of biology including survival, longevity, and development. However, the molecular links between autophagy and endocytosis are not well understood. Here, we report that BEC-1, the C. elegans ortholog of Atg6/Vps30/Beclin1, a key regulator of the autophagic machinery, also contributes to endosome function. In particular we identify a defect in retrograde transport from endosomes to the Golgi in bec-1 mutants. MIG-14/Wntless is normally recycled from endosomes to the Golgi through the action of the retromer complex and its associated factor RME-8. Lack of retromer or RME-8 activity results in the aberrant transport of MIG-14/Wntless to the lysosome where it is degraded. Similarly, we find that lack of bec-1 also results in mislocalization and degradation of MIG-14::GFP, reduced levels of RME-8 on endosomal membranes, and the accumulation of morphologically abnormal endosomes. A similar phenotype was observed in animals treated with dsRNA against vps-34. We further identify a requirement for BEC-1 in the clearance of apoptotic corpses in the hermaphrodite gonad, suggesting a role for BEC-1 in phagosome maturation, a process that appears to depend upon retrograde transport. In addition, autophagy genes may also be required for cell corpse clearance, as we find that RNAi against atg-18 or unc-51 also results in a lack of cell corpse clearance.     

The Atg6/Vps30/Beclin 1 ortholog BEC-1 mediates endocytic retrograde transport in addition to autophagy in C. elegans

Autophagy and endocytosis are dynamic and tightly regulated processes that contribute to many fundamental aspects of biology including survival, longevity, and development. However, the molecular links between autophagy and endocytosis are not well understood. Here, we report that BEC-1, the C. elegans ortholog of Atg6/Vps30/Beclin1, a key regulator of the autophagic machinery, also contributes to endosome function. In particular we identify a defect in retrograde transport from endosomes to the Golgi in bec-1 mutants. MIG-14/Wntless is normally recycled from endosomes to the Golgi through the action of the retromer complex and its associated factor RME-8. Lack of retromer or RME-8 activity results in the aberrant transport of MIG-14/Wntless to the lysosome where it is degraded. Similarly, we find that lack of bec-1 also results in mislocalization and degradation of MIG-14::GFP, reduced levels of RME-8 on endosomal membranes, and the accumulation of morphologically abnormal endosomes. A similar phenotype was observed in animals treated with dsRNA against vps-34. We further identify a requirement for BEC-1 in the clearance of apoptotic corpses in the hermaphrodite gonad, suggesting a role for BEC-1 in phagosome maturation, a process that appears to depend upon retrograde transport. In addition, autophagy genes may also be required for cell corpse clearance, as we find that RNAi against atg-18 or unc-51 also results in a lack of cell corpse clearance.     

Lysosomal calcium regulates autophagy

Recent evidence has indicated that the lysosome is able to act as a signaling organelle that senses nutrient availability and generates an adaptive response that is important for cellular homeostasis. We recently discovered another example of lysosomal signaling where lysosomal calcium release activates the master autophagy regulator TFEB via the phosphatase calcineurin.     

ER-mitochondria signaling regulates autophagy

The endoplasmic reticulum (ER) and mitochondria form tight functional contacts that regulate several key cellular processes. The formation of these contacts involves “tethering proteins” that function to recruit regions of ER to mitochondria. The integral ER protein VAPB (VAMP associated protein B and C) binds to the outer mitochondrial membrane protein, RMDN3/PTPIP51 (regulator of microtubule dynamics 3) to form one such set of tethers. Recently, we showed that the VAPB-RMDN3 tethers regulate macroautophagy/autophagy. Small interfering RNA (siRNA) knockdown of VAPB or RMDN3 to loosen ER-mitochondria contacts stimulates autophagosome formation, whereas overexpression of VAPB or RMDN3 to tighten contacts inhibit their formation. Artificial tethering of ER and mitochondria via expression of a synthetic linker protein also reduces autophagy and this artificial tether rescues the effects of VAPB- or RMDN3-targeted siRNA loss on autophagosome formation. Finally, our studies revealed that the modulatory effects of ER-mitochondria contacts on autophagy involve their role in mediating ITPR (inositol 1,4,5-trisphosphate receptor) delivery of Ca²⁺ from ER stores to mitochondria.     

Rab15 differentially regulates early endocytic trafficking

Rab GTPases play an important regulatory role in early endocytosis. We recently demonstrated that epitope-tagged Rab15 (HArab15) co-localizes with Rab4, -5, and -11 on early endosomal membranes in CHO cells (Zuk, P. A., and Elferink, L. A. (1999) J. Biol. Chem. 274, 22303-22312). To characterize the role of Rab15 in endocytosis, we prepared functional mutants of HArab15 and examined their effects on early endocytic trafficking. Wild-type HArab15 and its constitutively active, GTP-bound mutant (Q67L) reduce fluid phase and receptor-mediated endocytosis without affecting the rate of recycling from early endosomal compartments. Inhibition of early endocytosis appears to be due to a reduction in the rate of homotypic early endosome fusion. Conversely, mutations that constitutively inactivate HArab15 stimulate early endocytosis and the homotypic fusion of early endosomes in vitro. Unlike active forms of HArab15, constitutively inactive HArab15 mutants also affect recycling from early endosomal compartments. Moreover, the two constitutively inactive mutants, GDP-bound HArab15-T22N and the non-nucleotide binding mutant HArab15-N121I, differentially regulate the transit of fluid phase and receptor-mediated endocytic tracers through early/sorting endosomes. Together, these data suggest that HArab15 may counteract the reported stimulatory effect of Rab5 on early endocytosis. Consistent with this, overexpression of constitutively active HArab15-Q67L attenuates Rab5-stimulated endocytosis, whereas Rab5-stimulated endocytosis is augmented in cells overexpressing a constitutively inactive HArab15 mutant defective in guanine nucleotide binding (N121I). Our data indicate that HArab15 differentially regulates distinct steps in membrane trafficking through early/sorting and pericentriolar recycling endosomes.     

Vps9p CUE domain ubiquitin binding is required for efficient endocytic protein traffic

Rab5 GTPases are key regulators of protein trafficking through the early stages of the endocytic pathway. The yeast Rab5 ortholog Vps21p is activated by its guanine nucleotide exchange factor Vps9p. Here we show that Vps9p binds ubiquitin and that the CUE domain is necessary and sufficient for this interaction. Vps9p ubiquitin binding is required for efficient endocytosis of Ste3p but not for the delivery of the biosynthetic cargo carboxypeptidase Y to the vacuole. In addition, Vps9p is itself monoubiquitylated. Ubiquitylation is dependent on a functional CUE domain and Rsp5p, an E3 ligase that participates in cell surface receptor endocytosis. These findings define a new ubiquitin binding domain and implicate ubiquitin as a modulator of Vps9p function in the endocytic pathway.     

Endogenous HMGB1 regulates autophagy

Autophagy clears long-lived proteins and dysfunctional organelles and generates substrates for adenosine triphosphate production during periods of starvation and other types of cellular stress. Here we show that high mobility group box 1 (HMGB1), a chromatin-associated nuclear protein and extracellular damage-associated molecular pattern molecule, is a critical regulator of autophagy. Stimuli that enhance reactive oxygen species promote cytosolic translocation of HMGB1 and thereby enhance autophagic flux. HMGB1 directly interacts with the autophagy protein Beclin1 displacing Bcl-2. Mutation of cysteine 106 (C106), but not the vicinal C23 and C45, of HMGB1 promotes cytosolic localization and sustained autophagy. Pharmacological inhibition of HMGB1 cytoplasmic translocation by agents such as ethyl pyruvate limits starvation-induced autophagy. Moreover, the intramolecular disulfide bridge (C23/45) of HMGB1 is required for binding to Beclin1 and sustaining autophagy. Thus, endogenous HMGB1 is a critical pro-autophagic protein that enhances cell survival and limits programmed apoptotic cell death.     

An executioner caspase regulates autophagy

The relationships between autophagy and cell death are complex and still not well understood. To advance our understanding of the molecular connections between autophagy and apoptosis, we performed an RNAi-based screen of Drosophila melanogaster apoptosis-related genes for their ability to enhance or suppress starvation-induced autophagy. We discovered that six apoptosis-related genes, Dcp-1, hid, bruce, buffy, debcl and p53 as well as Ras/Raf/MAPK signaling pathway components play a role in autophagy regulation in Drosophila cultured cells. Our study also provides the first in vivo evidence that the effector caspase Dcp-1 and IAP protein Bruce regulate both autophagy and starvation-induced cell death at two nutrient status checkpoints, germarium and mid-oogenesis, in the Drosophila ovary. Analysis of degenerating mid-stage egg chambers in DmAtg1 and DmAtg7 mutants reveal a reduction in TUNEL staining though DNA condensation appears unaffected. Based on these and previous findings, we propose here a putative molecular pathway that might regulate the sensitivity threshold of apoptotic and autophagic responses. We also discuss multiple interpretations of the Atg mutant egg chamber TUNEL phenotype that are consistent with a possible role for autophagy in either suppressing or enhancing the efficiency of cell degradation and/or promoting cell clearance associated with the death process.     

Caenorhabditis elegans num-1 negatively regulates endocytic recycling

Much of the material taken into cells by endocytosis is rapidly returned to the plasma membrane by the endocytic recycling pathway. Although recycling is vital for the correct localization of cell membrane receptors and lipids, the molecular mechanisms that regulate recycling are only partially understood. Here we show that in Caenorhabditis elegans endocytic recycling is inhibited by NUM-1A, the nematode Numb homolog. NUM-1AGFP fusion protein is localized to the baso-lateral surfaces of many polarized epithelial cells, including the hypodermis and the intestine. We show that increased NUM-1A levels cause morphological defects in these cells similar to those caused by loss-of-function mutations in rme-1, a positive regulator of recycling in both C. elegans and mammals. We describe the isolation of worms lacking num-1A activity and show that, consistent with a model in which NUM-1A negatively regulates recycling in the intestine, loss of num-1A function bypasses the requirement for RME-1. Genetic epistasis analysis with rab-10, which is required at an early part of the recycling pathway, suggests that loss of num-1A function does not affect the uptake of material by endocytosis but rather inhibits baso-lateral recycling downstream of rab-10.     

The RCP-Rab11 complex regulates endocytic protein sorting

Rab 11 GTPase is an important regulator of endocytic membrane traffic. Recently, we and others have identified a novel family of Rab11 binding proteins, known as Rab11-family interacting proteins (FIPs). One of the family members, Rab coupling protein (RCP), was identified as a protein binding to both Rab4 and Rab11 GTPases. RCP was therefore suggested to serve a dual function as Rab4 and Rab11 binding protein. In this study, we characterized the cellular functions of RCP and mapped its interactions with Rab4 and Rab11. Our data show that RCP interacts only weakly with Rab4 in vitro and does not play the role of coupling Rab11 and Rab4 in vivo. Furthermore, our data indicate that the RCP-Rab11 complex regulates the sorting of transferrin receptors from the degradative to the recycling pathway. We therefore propose that RCP functions primarily as a Rab11 binding protein that regulates protein sorting in tubular endosomes.     

Rabankyrin-5 interacts with EHD1 and Vps26 to regulate endocytic trafficking and retromer function

Rabankyrin-5 (Rank-5) has been implicated as an effector of the small GTPase Rab5 and plays an important role in macropinocytosis. We have now identified Rank-5 as an interaction partner for the recycling regulatory protein, Eps15 homology domain 1 (EHD1). We have demonstrated this interaction by glutathione S-transferase-pulldown, yeast two-hybrid assay, isothermal calorimetry and co-immunoprecipitation, and found that the binding occurs between the EH domain of EHD1 and the NPFED motif of Rank-5. Similar to EHD1, we found that Rank-5 colocalizes and interacts with components of the retromer complex such as vacuolar protein sorting 26 (Vps26), suggesting a role for Rank-5 in retromer-based transport. Indeed, depletion of Rank-5 causes mislocalization of Vps26 and affects both the retrieval of mannose 6-phosphate receptor transport to the Golgi from endosomes and biosynthetic transport. Moreover, Rank-5 is required for normal retromer distribution, as overexpression of a wild-type Rank-5-small interfering RNA-resistant construct rescues retromer mislocalization. Finally, we show that depletion of either Rank-5 or EHD1 impairs secretion of vesicular stomatitis virus glycoprotein. Overall, our data identify a new interaction between Rank-5 and EHD1, and novel endocytic regulatory roles that include retromer-based transport and secretion.     

A novel TIP30 protein complex regulates EGF receptor signaling and endocytic degradation

Activated epidermal growth factor receptor (EGFR) continues to signal in the early endosome, but how this signaling process is regulated is less well understood. Here we describe a protein complex consisting of TIP30, endophilin B1, and acyl-CoA synthetase long chain family member 4 (ACSL4) that interacts with Rab5a and regulates EGFR endocytosis and signaling. These proteins are required for the proper endocytic trafficking of EGF-EGFR. Knockdown of TIP30, ACSL4, endophilin B1, or Rab5a in human liver cancer cells or genetic knock-out of Tip30 in mouse primary hepatocytes results in the trapping of EGF-EGFR complexes in early endosomes, leading to delayed EGFR degradation and prolonged EGFR signaling. Furthermore, we show that Rab5a colocalizes with vacuolar (H(+))-ATPases (V-ATPases) on transport vesicles. The TIP30 complex facilitates trafficking of Rab5a and V-ATPases to EEA1-positive endosomes in response to EGF. Together, these results suggest that this TIP30 complex regulates EGFR endocytosis by facilitating the transport of V-ATPases from trans-Golgi network to early endosomes.     

Phosphatidylethanolamine positively regulates autophagy and longevity

Autophagy is a cellular recycling program that retards ageing by efficiently eliminating damaged and potentially harmful organelles and intracellular protein aggregates. Here, we show that the abundance of phosphatidylethanolamine (PE) positively regulates autophagy. Reduction of intracellular PE levels by knocking out either of the two yeast phosphatidylserine decarboxylases (PSD) accelerated chronological ageing-associated production of reactive oxygen species and death. Conversely, the artificial increase of intracellular PE levels, by provision of its precursor ethanolamine or by overexpression of the PE-generating enzyme Psd1, significantly increased autophagic flux, both in yeast and in mammalian cell culture. Importantly administration of ethanolamine was sufficient to extend the lifespan of yeast (Saccharomyces cerevisiae), mammalian cells (U2OS, H4) and flies (Drosophila melanogaster). We thus postulate that the availability of PE may constitute a bottleneck for functional autophagy and that organismal life or healthspan could be positively influenced by the consumption of ethanolamine-rich food.Phosphatidylethanolamine (PE) is a phospholipid found in all living organisms. Together with phosphatidylcholine (PC), phosphatidylserine (PS) and phosphatidylinositol (PI), PE represents the backbone of most biological membranes. PE is the second-most abundant phospholipid in mammalian membranes ranging from 20 to 50%.¹ In yeast, PE is essential for growth and is generated through four different enzymatic pathways:² PE can be produced by decarboxylation of PS, as a first option at the mitochondrial membrane via phosphatidylserine decarboxylase 1 (Psd1)^{3, 4} or, as a second, option at the Golgi and vacuolar membranes through phosphatidylserine decarboxylase 2 (Psd2).⁵ As a third possibility, PE can be produced from actively retrieved extracellular ethanolamine,^{6, 7} which is cytidine 5''-diphosphate-activated⁸ and then coupled to diacylglycerol to generate PE.⁹ The fourth, scarcely employed PE-generating pathway is based on the lysophospholipid acylation of lyso-PE. Importantly, PE does not spontaneously assemble in bilayers and rather incorporates into curved structures, such as the inverted hexagonal phase.¹⁰ The physiological function of non-bilayer lipids in membranes is considered to reside in their interaction with membrane proteins via the membrane lateral pressure¹⁰ and membrane tethering and fusion processes, which are relevant for autophagy.¹¹The term ‘autophagy'' describes a degradation process affecting intracellular components (for a review see, 12 13) which as an important cytoprotective mechanism, is closely linked to ageing. Autophagy mainly differs from the proteasomal pathway, the other major cellular degradation mechanism, in two aspects. First, autophagy can degrade large particles or whole organelles and second, the final degradation occurs in the lysosome/vacuole and not at the proteasome. Prior to the actual degradation, the cargo is gathered in autophagic particles, which are surrounded by a characteristic double-membrane. However, the origin of these autophagosomal membranes is still controversial and might actually depend on the mode of autophagy induction.^{14, 15} Among the discussed membrane sources are the Golgi apparatus, the endosplamic reticulum (ER) or the mitochondrion-associated membrane, which is formed at the interface between the ER.¹⁶ In higher eukaryotes autophagic membranes are enriched in PE with a high degree of unsaturation,¹⁷ similarly to the PE species found in mitochondria.^{14, 18} Moreover, the pre-autophagosomal structure or phagophore assembly site (PAS), which appears at the very beginning of autophagosome formation, already harbours Atg9, an autophagy-related transmembrane protein that shuttles between mitochondria and the PAS structure in yeast.¹⁹Importantly, PE also functions as an anchor to autophagosomal membranes for the autophagy-related protein Atg8 in yeast²⁰ and its mammalian orthologue LC3.^{21, 22} This PE anchor is provided to LC3/Atg8 post-translationally in a process called lipidation. First, LC3/Atg8 is carboxy-terminally cleaved by proteases from the Atg4 family.^{23, 24} Subsequently, the remaining C-terminal glycine is coupled to PE in a series of ubiquitination-like reactions involving diverse Atg-proteins.^{20, 25, 26, 27} In vitro, Atg8-PE causes hemifusion of vesicles, which argues for its potential role in autophagosomal phagophore expansion.^{11, 28} Consistently, semisynthetic LC3-PE has recently been described to stimulate membrane tethering and fusion.²⁹ We thus reasoned that the overall abundance of PE might be critical for PE-lipidation of LC3/Atg8 and could thus regulate autophagosomal membrane formation. Therefore, we tested whether increasing cellular PE levels might have an impact on autophagy and lifespan regulation.Here, we report that knock-out of PSD1 or PSD2 shortens the chronological lifespan of S. cerevisiae, whereas PSD1-overexpression enhances the autophagic capacity and increases longevity. Furthermore, external administration of ethanolamine increases endogenous PE levels, enhances autophagic flux and extends the lifespan of yeast, mammalian cells in culture and flies (Drosophila melanogaster).     

Dengue virus-induced autophagy regulates lipid metabolism

Autophagy influences numerous cellular processes, including innate and adaptive immunity against intracellular pathogens. However, some viruses, including dengue virus (DENV), usurp autophagy to enhance their replication. The mechanism for a positive role of autophagy in DENV infection is unclear. We present data that DENV induction of autophagy regulates cellular lipid metabolism. DENV infection leads to an autophagy-dependent processing of lipid droplets and triglycerides to release free fatty acids. This results in an increase in cellular β-oxidation, which generates ATP. These processes are required for efficient DENV replication. Importantly, exogenous fatty acids can supplant the requirement of autophagy in DENV replication. These results define a role for autophagy in DENV infection and provide a mechanism by which viruses can alter cellular lipid metabolism to promote their replication.     

The Vps4p AAA ATPase regulates membrane association of a Vps protein complex required for normal endosome function.

Vps4p is an AAA-type ATPase required for efficient transport of biosynthetic and endocytic cargo from an endosome to the lysosome-like vacuole of Saccharomyces cerevisiae. Vps4p mutants that do not bind ATP or are defective in ATP hydrolysis were characterized both in vivo and in vitro. The nucleotide-free or ADP-bound form of Vps4p existed as a dimer, whereas in the ATP-locked state, Vps4p dimers assembled into a decameric complex. This suggests that ATP hydrolysis drives a cycle of association and dissociation of Vps4p dimers/decamers. Nucleotide binding also regulated the association of Vps4p with an endosomal compartment in vivo. This membrane association required the N-terminal coiled-coil motif of Vps4p, but deletion of the coiled-coil domain did not affect ATPase activity or oligomeric assembly of the protein. Membrane association of two previously uncharacterized class E Vps proteins, Vps24p and Vps32p/Snf7p, was also affected by mutations in VPS4. Upon inactivation of a temperature-conditional vps4 mutant, Vps24p and Vps32p/Snf7p rapidly accumulated in a large membrane-bound complex. Immunofluorescence indicated that both proteins function with Vps4p at a common endosomal compartment. Together, the data suggest that the Vps4 ATPase catalyzes the release (uncoating) of an endosomal membrane-associated class E protein complex(es) required for normal morphology and sorting activity of the endosome.