Recruitment of the endosomal WASH complex is mediated by the extended ‘tail’ of Fam21 binding to the retromer protein Vps35

2012 ◽  
Vol 442 (1) ◽  
pp. 209-220 ◽  
Author(s):  
Michael E. Harbour ◽  
Sophia Y. Breusegem ◽  
Matthew N. J. Seaman
Keyword(s):  
Membrane Proteins ◽  
Protein Sorting ◽  
Complex Interaction ◽  
Membrane Association ◽  
Tail Domain ◽  
Fk506 Binding Protein ◽  
Retromer Complex ◽  
Multimeric Protein ◽  
Vacuolar Protein ◽  
Necessary And Sufficient

The retromer complex is a conserved endosomal protein sorting complex that sorts membrane proteins into nascent endosomal tubules. The recognition of membrane proteins is mediated by the cargo-selective retromer complex, a stable trimer of the Vps35 (vacuolar protein sorting 35), Vps29 and Vps26 proteins. We have recently reported that the cargo-selective retromer complex associates with the WASH (Wiskott–Aldrich syndrome homologue) complex, a multimeric protein complex that regulates tubule dynamics at endosomes. In the present study, we show that the retromer–WASH complex interaction occurs through the long unstructured ‘tail’ domain of the WASH complex–Fam21 protein binding to Vps35, an interaction that is necessary and sufficient to target the WASH complex to endosomes. The Fam21-tail also binds to FKBP15 (FK506-binding protein 15), a protein associated with ulcerative colitis, to mediate the membrane association of FKBP15. Elevated Fam21-tail expression inhibits the association of the WASH complex with retromer, resulting in increased cytoplasmic WASH complex. Additionally, overexpression of the Fam21-tail results in cell-spreading defects, implicating the activity of the WASH complex in regulating the mobilization of membrane into the endosome-to-cell surface pathway.

scholarly journals Identification of a conserved motif required for Vps35p/Vps26p interaction and assembly of the retromer complex

2007 ◽  
Vol 408 (2) ◽  
pp. 287-295 ◽  
Author(s):  
Suzanne Gokool ◽  
Daniel Tattersall ◽  
Jonathan V. Reddy ◽  
Matthew N. J. Seaman
Keyword(s):  
Protein Sorting ◽  
Membrane Association ◽  
Vacuolar Protein Sorting ◽  
Conserved Motif ◽  
Endosomal Membrane ◽  
Retromer Complex ◽  
Complex Mutation ◽  
Cargo Proteins ◽  
Conserved Region ◽  
Vacuolar Protein

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.

scholarly journals Vacuolar protein sorting receptor in Schizosaccharomyces pombe

Microbiology ◽  
2006 ◽  
Vol 152 (5) ◽  
pp. 1523-1532 ◽  
Author(s):  
Tomoko Iwaki ◽  
Akira Hosomi ◽  
Sanae Tokudomi ◽  
Yoko Kusunoki ◽  
Yasuko Fujita ◽  
...  
Keyword(s):  
Schizosaccharomyces Pombe ◽  
Mammalian Cells ◽  
Fluorescent Protein ◽  
Transmembrane Protein ◽  
Protein Sorting ◽  
Retrograde Transport ◽  
Carboxypeptidase Y ◽  
Type I ◽  
Tail Domain ◽  
Vacuolar Protein

The mechanism by which soluble proteins, such as carboxypeptidase Y, reach the vacuole in Saccharomyces cerevisiae is very similar to the mechanism of lysosomal protein sorting in mammalian cells. Vps10p is a receptor for transport of soluble vacuolar proteins in S. cerevisiae. vps10 +, a gene encoding a homologue of S. cerevisiae PEP1/VPS10, has been identified and deleted from the fission yeast Schizosaccharomyces pombe. Deletion of the vps10 + gene resulted in missorting and secretion of Sch. pombe vacuolar carboxypeptidase Cpy1p, indicating that it is required for targeting Cpy1p to the vacuole. Sch. pombe Vps10p (SpVps10p) is a type I transmembrane protein and its C-terminal cytoplasmic tail domain is essential for Cpy1p transport to the vacuole. Cells expressing green fluorescent protein-tagged SpVps10p produced a punctate pattern of fluorescence, indicating that SpVps10p was largely localized in the Golgi compartment. In addition, Sch. pombe vps26 +, vps29 + and vps35 +, encoding homologues of the S. cerevisiae retromer components VPS26, VPS29 and VPS35, were identified and deleted. Fluorescence microscopy demonstrated that SpVps10p mislocalized to the vacuolar membrane in these mutants. These results indicate that the vps26 +, vps29 + and vps35 + gene products are required for retrograde transport of SpVps10p from the prevacuolar compartment back to the Golgi in Sch. pombe cells.

scholarly journals SEC18/NSF-independent, protein-sorting pathway from the yeast cortical ER to the plasma membrane

2005 ◽  
Vol 169 (4) ◽  
pp. 613-622 ◽  
Author(s):  
Christoph Jüschke ◽  
Andrea Wächter ◽  
Blanche Schwappach ◽  
Matthias Seedorf
Keyword(s):  
Plasma Membrane ◽  
Membrane Proteins ◽  
Protein Sorting ◽  
Temperature Sensitive ◽  
Cell Periphery ◽  
Yeast Protein ◽  
Local Synthesis ◽  
Sorting Signals ◽  
Necessary And Sufficient ◽  
Independent Protein

Classic studies of temperature-sensitive secretory (sec) mutants have demonstrated that secreted and plasma membrane proteins follow a common SEC pathway via the endoplasmic reticulum (ER), Golgi apparatus, and secretory vesicles to the cell periphery. The yeast protein Ist2p, which is synthesized from a localized mRNA, travels from the ER to the plasma membrane via a novel route that operates independently of the formation of coat protein complex II–coated vesicles. In this study, we show that the COOH-terminal domain of Ist2p is necessary and sufficient to mediate SEC18-independent sorting when it is positioned at the COOH terminus of different integral membrane proteins and exposed to the cytoplasm. This domain functions as a dominant plasma membrane localization determinant that overrides other protein sorting signals. Based on these observations, we suggest a local synthesis of Ist2p at cortical ER sites, from where the protein is sorted by a novel mechanism to the plasma membrane.

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

1999 ◽  
Vol 10 (6) ◽  
pp. 1873-1889 ◽  
Author(s):  
Gregory G. Tall ◽  
Hiroko Hama ◽  
Daryll B. DeWald ◽  
Bruce F. Horazdovsky
Keyword(s):  
Protein Sorting ◽  
Specific Interaction ◽  
Snare Complex ◽  
Membrane Association ◽  
Rab Proteins ◽  
Phosphate Binding ◽  
Vacuolar Protein Sorting ◽  
Rab Protein ◽  
Vacuolar Protein ◽  
Phosphatidylinositol 3

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 betweenVPS21 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 ofVPS34. 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.

scholarly journals An Interorganellar Bidding War: Vps13 Localization by Competitive Organelle-Specific Adaptors

Contact ◽  
2018 ◽  
Vol 1 ◽  
pp. 251525641881462
Author(s):  
Samantha K. Dziurdzik ◽  
Björn D.M. Bean ◽  
Elizabeth Conibear
Keyword(s):  
Endoplasmic Reticulum ◽  
Recent Work ◽  
Protein Sorting ◽  
Repeat Region ◽  
Vacuolar Protein Sorting ◽  
Membrane Contact Sites ◽  
Contact Sites ◽  
Prospore Membrane ◽  
Membrane Contact ◽  
Vacuolar Protein

Membrane contact sites are regulated through the controlled recruitment of constituent proteins. Yeast vacuolar protein sorting 13 (Vps13) dynamically localizes to membrane contact sites at endosomes, vacuoles, mitochondria, and the endoplasmic reticulum under different cellular conditions and is recruited to the prospore membrane during meiosis. Prior to our recent work, the mechanism for localization at contact sites was largely unknown. We identified Ypt35 as a novel Vps13 adaptor for endosomes and the nucleus-vacuole junction. Furthermore, we discovered a conserved recruitment motif in Ypt35 and found related motifs in the prospore membrane and mitochondrial adaptors, Spo71 and Mcp1, respectively. All three adaptors compete for binding to a six-repeat region of Vps13, suggesting adaptor competition regulates Vps13 localization. Here, we summarize and discuss the implications of our work, highlighting key outstanding questions.

scholarly journals The abundance and activation of mTORC1 regulators in skeletal muscle of neonatal pigs are modulated by insulin, amino acids, and age

2010 ◽  
Vol 109 (5) ◽  
pp. 1448-1454 ◽  
Author(s):  
Agus Suryawan ◽  
Teresa A. Davis
Keyword(s):  
Skeletal Muscle ◽  
Mammalian Target Of Rapamycin ◽  
Developmentally Regulated ◽  
Fk506 Binding Protein ◽  
Neonatal Pigs ◽  
Cell Culture Systems ◽  
Mtorc1 Signaling ◽  
Phospholipase D1 ◽  
Vacuolar Protein

Mammalian target of rapamycin complex 1 (mTORC1) signaling is crucial for the regulation of protein synthesis. Most of known mTORC1 regulators have been isolated and characterized using cell culture systems, and the physiological roles of these regulators have not been fully tested in vivo. Previously we demonstrated that the insulin (INS) and amino acid (AA)-induced activation of mTORC1 is developmentally regulated in skeletal muscle (Suryawan A et al. Am J Physiol Endocrinol Metab 293: E1597–E1605, 2007). The present study aimed to characterize in more detail the effects of the postprandial rise in INS and AA on the activation and abundance of mTORC1 regulators in muscle and how this is modified by development. Overnight fasted 6- and 26-day-old pigs were studied during 1) euinsulinemic-euglycemic-euaminoacidemic conditions (control), 2) euinsulinemic-euglycemic-hyperaminoacidemic clamps (AA), and 3) hyperinsulinemic-euglycemic-euaminoacidemic clamps (INS). INS, but not AA, enhanced the PRAS40 phosphorylation, and this effect was greater in 6- than in 26-day old pigs. Phospholipase D1 (PLD1) abundance and phosphorylation, and the association of PLD1 with Ras homolog enriched in brain (Rheb), were greater in the younger pigs. Neither INS, AA, nor age altered the abundance of Rheb, vacuolar protein sorting 34 (Vps34), or FK506-binding protein 38 (FKBP38). Although INS and AA had no effect, the abundance of ras-related GTP binding B (RagB) and the association of RagB with Raptor were greater in 6- than in 26-day-old pigs. Neither INS, AA, nor age altered AMPK-induced phosphorylation of Raptor. Our results suggest that the enhanced activation of mTORC1 in muscle of neonatal pigs is in part due to regulation by PRAS40, PLD1, and the Rag GTPases.

The yeast VPS5/GRD2 gene encodes a sorting nexin-1-like protein required for localizing membrane proteins to the late Golgi

1997 ◽  
Vol 110 (9) ◽  
pp. 1063-1072 ◽  
Author(s):  
S.F. Nothwehr ◽  
A.E. Hindes
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Protein Localization ◽  
Endocytic Pathway ◽  
Yeast Cells ◽  
Carboxypeptidase Y ◽  
Golgi Membrane ◽  
Sorting Nexin ◽  
Vacuolar Protein ◽  
Sorting Nexin 1

Genetic analysis of late Golgi membrane protein localization in Saccharomyces cerevisiae has uncovered a large number of genes (called GRD) that are required for retention of A-ALP, a model late Golgi membrane protein. Here we describe one of the GRD genes, VPSS/GRD2, that encodes a hydrophilic protein similar to human sorting nexin-1, a protein involved in trafficking of the epidermal growth factor receptor. In yeast cells containing a vps5 null mutation the late Golgi membrane proteins A-ALP and Kex2p were rapidly mislocalized to the vacuolar membrane. A-ALP was delivered to the vacuole in vps5 mutants in a manner independent of a block in the early endocytic pathway. vps5 null mutants also exhibited defects in both vacuolar morphology and in sorting of a soluble vacuolar protein, carboxypeptidase Y. The latter defect is apparently due to an inability to localize the carboxypeptidase Y sorting receptor, Vps10p, to the Golgi since it is rapidly degraded in the vacuole in vps5 mutants. Fractionation studies indicate that Vps5p is distributed between a free cytosolic pool and a particulate fraction containing Golgi, transport vesicles, and possibly endosomes, but lacking vacuolar membranes. Immunofluorescence microscopy experiments show that the membrane-associated pool of Vps5p localizes to an endosome-like organelle that accumulates in the class E vps27 mutant. These results support a model in which Vps5p is required for retrieval of membrane proteins from a prevacuolar/late endosomal compartment back to the late Golgi apparatus.

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