Retromer function in endosome-to-Golgi retrograde transport is regulated by the yeast Vps34 PtdIns 3-kinase (original) (raw)
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The Journal of cell biology, 1995
A membrane-associated complex composed of the Vps15 protein kinase and the Vps34 phosphatidylinositol 3-kinase (PtdIns 3-kinase) is essential for the delivery of proteins to the yeast vacuole. An active Vps15p is required for the recruitment of Vps34p to the membrane and subsequent stimulation of Vps34p PtdIns 3-kinase activity. Consistent with this, mutations altering highly conserved residues in the lipid kinase domain of Vps34p lead to a dominant-negative phenotype resulting from titration of activating Vps15 proteins. In contrast, catalytically inactive Vps15p mutants do not produce a dominant mutant phenotype because they are unable to associate with Vps34p in a wild-type manner. These data indicate that an intact Vps15p protein kinase domain is necessary for the association with and activation of Vps34p, and they demonstrate that a functional Vps15p-Vps34p complex is absolutely required for the efficient delivery of proteins to the vacuole. Analysis of a temperature-conditiona...
Biochemical Journal, 2007
The retromer complex is a conserved cytoplasmic coat complex that mediates the endosome-to-Golgi retrieval of vacuole/lysosome hydrolase receptors in yeast and mammals. The recognition of cargo proteins by the retromer is performed by the Vps35p/VPS35 (where Vps is vacuolar protein sorting) component, which together with Vps26p/VPS26 and Vps29p/VPS29, forms the cargo-selective subcomplex. In this report, we have identified a highly-conserved region of Vps35p/VPS35 that is essential for the interaction with Vps26p/VPS26 and for assembly of the retromer complex. Mutation of residues within the conserved region results in Vps35p/VPS35 mutants, which cannot bind to Vps26p/VPS26 and are not efficiently targeted to the endosomal membrane. These data implicate Vps26p/VPS26 in regulating Vps35p/VPS35 membrane association and therefore suggest a role for Vps26p/VPS26 in cargo recognition.
2003
Autophagy, pexophagy, and the Cvt pathway are processes that deliver hydrolytic enzymes and substrates to the yeast vacuole/lysosome via double-membrane cytosolic vesicles. Whereas these pathways operate under different nutritional conditions, they all employ common machinery with only a few specific factors assisting in the choice of the delivery program and the membrane source for the sequestering vesicle. We found that the YKR020w gene product is essential for Cvt vesicle formation but not for pexophagy or induction of autophagy. Autophagosomes in the ykr020wΔ mutant, however, have a reduced size. We demonstrate that Ykr020 is a subunit of the Vps fiftythree tethering complex, composed of Vps52, Vps53, and Vps54, which is required for retrograde traffic from the early endosome back to the late Golgi, and for this reason we named it Vps51. This complex participates in a fusion event together with Tlg1 and Tlg2, two SNAREs also shown to be necessary for Cvt vesicle assembly. In particular, those factors are essential to correctly target the prApe1-Cvt19-Cvt9 complex to the preautophagosomal structure, the site of Cvt vesicle formation. Autophagy is a catabolic process conserved among yeast, plants, and animal cells that permits the cell to eliminate unwanted or unnecessary proteins and organelles and to recycle the components for reuse (1,2). The organellar turnover is exclusively accomplished in the lysosome/vacuole lumen by a wide range of hydrolases capable of breaking down all cellular constituents (1,2). Autophagy plays an essential role during normal physiological processes such as starvation, cellular differentiation, cell death, and aging, but also in preventing some types of cellular dysfunction including cancer (2).
Journal of Biological Chemistry, 2003
Phosphatidylinositol 3,5-bisphosphate is a membrane lipid found in all eukaryotes so far studied but downstream effector proteins of this lipid have yet to be identified. Here we report the use of cDNA phage libraries in conjunction with synthetic biotinylated derivatives of phosphatidylinositol 3,5-bisphosphate in the identification of a mammalian phosphatidylinositol 3,5-bisphosphate-binding protein, mVps24p. This protein is orthologous to the Saccharomyces cerevisiae protein, Vps24p, a class-E vacuolar protein-sorting protein. Using in vitro liposome binding and competition assays, we demonstrate that mVps24p selectively binds to phosphatidylinositol 3,5-bisphosphate and phosphatidylinositol 3,4bisphosphate in preference to other phosphoinositides tested. When expressed in cultured mammalian cells, full-length mVps24p is cytosolic. However, when cells expressing the full-length mVps24p are co-transfected with a mutated form of mVps4p (which is defective in ATP hydrolysis), or when a N-terminal construct of mVps24p is expressed, the class-E cellular phenotype with swollen vacuoles is induced and mVps24p is membrane-associated. Furthermore, the accumulation of the N-terminal mVps24p construct on the swollen endosomal membranes is abrogated when phosphatidylinositol 3,5-bisphosphate synthesis is blocked with wortmannin. These data provide the first direct link between phosphatidylinositol 3,5-bisphosphate and the protein machinery involved in the production of the class-E cellular phenotype. We hypothesize that accumulation of Vps24 on membranes occurs when membrane association (dependent on interaction of phosphatidylinositol 3,5-bisphosphate with the N-terminal domain of the protein) is uncoupled from membrane disassociation (driven by Vps4p).
The EMBO Journal, 1993
Communicated by D.Meyer 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. Overproduction of Vps34p suppresses the growth and vacuolar protein sorting defects associated with vpslS kinase domain mutants, suggesting that Vpsl5p and Vps34p functionally interact. Subceliular fractionation and sucrose density gradients indicate that Vpsl5p is responsible for the association of Vps34p with an intracellular membrane fraction. Chemical cross-linking and native immunoprecipitation experiments demonstrate that Vpsl5p 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 Vpsl5p-mediated protein phosphorylation event. We propose that Vpsl5p 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.
SOI3 was identified by a mutation, soi3-1, that suppressed a mutant trans-Golgi network (TGN) localization signal in the Kex2p cytosolic tail. SOI3, identical to RAV1, encodes a protein important for regulated assembly of vacuolar ATPase. Here, we show that Soi3/Rav1p is required for transport between the early endosome and the late endosome/prevacuolar compartment (PVC). By electron microscopy, soi3-1 mutants massively accumulated structures that resembled early endosomes. soi3⌬ mutants exhibited a kinetic delay in transfer of the endocytic tracer dye FM4-64, from the 14°C endocytic intermediate to the vacuole. The soi3⌬ mutation delayed vacuolar degradation but not internalization of the a-factor receptor Ste3p. By density gradient fractionation, Soi3/Rav1p associated as a peripheral protein with membranes of a density characteristic of early endosomes. The soi3 null mutation markedly reduced the rate of Kex2p transport from the TGN to the PVC but had no effect on vacuolar protein sorting or cycling of Vps10p. These results suggest that assembly of vacuolar ATPase at the early endosome is required for transport of both Ste3p and Kex2p from the early endosome to the PVC and support a model in which cycling through the early endosome is part of the normal itinerary of Kex2p and other TGN-resident proteins.
Nature Cell Biology, 2013
Endoplasmic reticulum (ER)-Golgi membrane transport and autophagy are intersecting trafficking pathways that are tightly regulated and crucial for homeostasis, development and disease. Here, we identify UVRAG, a beclin-1-binding autophagic factor, as a phosphatidylinositol-3-phosphate (PtdIns(3)P)-binding protein that depends on PtdIns(3)P for its ER localization. We further show that UVRAG interacts with RINT-1, and acts as an integral component of the RINT-1-containing ER tethering complex, which couples phosphoinositide metabolism to COPI-vesicle tethering. Displacement or knockdown of UVRAG profoundly disrupted COPI cargo transfer to the ER and Golgi integrity. Intriguingly, autophagy caused the dissociation of UVRAG from the ER tether, which in turn worked in concert with the Bif-1-beclin-1-PI(3)KC3 complex to mobilize Atg9 translocation for autophagosome formation. These findings identify a regulatory mechanism that coordinates Golgi-ER retrograde and autophagy-related vesicular trafficking events through physical and functional interactions between UVRAG, phosphoinositide and their regulatory factors, thereby ensuring spatiotemporal fidelity of membrane trafficking and maintenance of organelle homeostasis. Membrane trafficking is essential for organelle homeostasis and basal cellular functions. Given numerous intersecting trafficking pathways in cells, they must be coordinated in time and space for targeted cargo delivery. The organelles and vesicles are coded with distinct phosphoinositides, which serve as signalling and/or binding platforms for effectors to direct the membrane flow 1,2. PtdIns(3)P is an essential regulator of endocytic, phagocytic and autophagic trafficking 1,3. However, a recent study suggested that PtdIns(3)P is also required for ER-related functions 4. Using a high-performance liquid chromatography method, another report further demonstrated that the ER contains more PtdIns(3)P than previously expected 5. In support of this view, both the class III PtdIns(3)-OH kinase (PI(3)KC3) and the myotubularin-related protein 3 (MTMR3), a PtdIns(3)-phosphatase,
Global Analysis of Yeast Endosomal Transport Identifies the Vps55/68 Sorting Complex
Molecular Biology of The Cell, 2008
Endosomal transport is critical for cellular processes ranging from receptor downregulation and retroviral budding to the immune response. A full understanding of endosome sorting requires a comprehensive picture of the multiprotein complexes that orchestrate vesicle formation and fusion. Here, we use unsupervised, large-scale phenotypic analysis and a novel computational approach for the global identification of endosomal transport factors. This technique effectively identifies components of known and novel protein assemblies. We report the characterization of a previously undescribed endosome sorting complex that contains two well-conserved proteins with 4 predicted membrane-spanning domains. Vps55p and Vps68p form a complex that acts with or downstream of ESCRT function to regulate endosomal trafficking. Loss of Vps68p disrupts recycling to the TGN as well as onward trafficking to the vacuole without preventing the formation of lumenal vesicles within the MVB. Our results suggest the Vps55/68 complex mediates a novel, conserved step in the endosomal maturation process. 2 by guest on June 3, 2013 http://www.molbiolcell.org/ Downloaded from
The Journal of cell biology, 2017
Yeast VPS13 is the founding member of a eukaryotic gene family of growing interest in cell biology and medicine. Mutations in three of four human VPS13 genes cause autosomal recessive neurodegenerative or neurodevelopmental disease, making yeast Vps13p an important structural and functional model. Using cell-free reconstitution with purified Vps13p, we show that Vps13p is directly required both for transport from the trans-Golgi network (TGN) to the late endosome/prevacuolar compartment (PVC) and for TGN homotypic fusion. Vps13p must be in complex with the small calcium-binding protein Cdc31p to be active. Single-particle electron microscopic analysis of negatively stained Vps13p indicates that this 358-kD protein is folded into a compact rod-shaped density (20 × 4 nm) with a loop structure at one end with a circular opening ∼6 nm in diameter. Vps13p exhibits ATP-stimulated binding to yeast membranes and specific interactions with phosphatidic acid and phosphorylated forms of phosph...
Journal of Cell …, 2010
Vesicles formed by the COPI complex function in retrograde transport from the Golgi to the endoplasmic reticulum (ER). Phosphatidylinositol transfer protein b (PITPb), an essential protein that possesses phosphatidylinositol (PtdIns) and phosphatidylcholine (PtdCho) lipid transfer activity is known to localise to the Golgi and ER but its role in these membrane systems is not clear. To examine the function of PITPb at the Golgi-ER interface, RNA interference (RNAi) was used to knockdown PITPb protein expression in HeLa cells. Depletion of PITPb leads to a decrease in PtdIns(4)P levels, compaction of the Golgi complex and protection from brefeldin-Amediated dispersal to the ER. Using specific transport assays, we show that anterograde traffic is unaffected but that KDEL-receptordependent retrograde traffic is inhibited. This phenotype can be rescued by expression of wild-type PITPb but not by mutants defective in docking, PtdIns transfer and PtdCho transfer. These data demonstrate that the PtdIns and PtdCho exchange activity of PITPb is essential for COPI-mediated retrograde transport from the Golgi to the ER.