Identification of the Functional Domains of Yeast Sorting Nexins Vps5p and Vps17p

Seaman, Matthew N.; Williams, Hazel P.

doi:10.1091/mbc.02-05-0064

Cited by 99 publications

(111 citation statements)

References 32 publications

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“…this reflects characteristics of other parts of these sorting nexins-particularly their coiled-coil regions, which have been shown to drive homo-and hetero-oligomerization in other studies. This finding has been confirmed independently in a study of the Vps17p-Vps5p interaction (31). A notable class of preys consists of the Yip1 family of proteins (32).…”

Section: Resultsmentioning

confidence: 61%

The Phox Homology (PX) Domain Protein Interaction Network in Yeast

Vollert¹,

Uetz²

2004

Molecular & Cellular Proteomics

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Phosphoinositides (PIs)1 act as signaling molecules in a variety of cellular processes including vesicular trafficking and cell motility (1-3). PI signaling is mediated by proteins that contain one or several lipid-binding domains such as PH, FYVE, ENTH, ANTH, FERM, PHD, Tubby, and C2 domains, all of which have been reported to bind PIs. Recently, the PX domain was shown to represent another PI-binding domain (reviewed in Refs. 4 -7).Phox homology (PX) domains were originally identified as a common motif of about 120 amino acids found in the p40 phox and p47 phox subunits of the neutrophil NADPH oxidase (Phox) complex (8). This domain is conserved from yeast to human, and more than 400 examples have been annotated in sequence databases (e.g. SMART (9)). PX domain-containing proteins have been implicated in highly diverse functions such as cell signaling, vesicular trafficking, protein sorting, and lipid modification (reviewed in Refs. 10 and 11).PX domains show relatively little sequence conservation, yet their structure appears to be highly conserved. All published crystal structures exhibit a clearly defined PI-binding pocket (12-15). Although phosphatidylinositol-3-phosphate (PtdIns(3)P) is the primary target of PX domains, binding to phosphatidic acid, phosphatidylinositol-3,4-bisphosphate (PtdIns(3,4)P 2 ), phosphatidylinositol-3,5-bisphosphate (PtdIns-(3,5)P 2 ), phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P 2 ), and phosphatidylinositol-3,4,5-trisphosphate (PtdIns(3,4,5)P 3 ) has been reported as well (16,17).The yeast genome encodes 15 PX domain-containing proteins that are involved in vesicular trafficking, cell signaling, control of bud emergence and cell polarity, or are of largely unknown function (Table I). All yeast PX domains (except the sequence outlier Ypr097p) bind specifically to PtdIns(3)P with different affinities (18).Although most PX domains contain additional domains-or at least additional protein sequences-two yeast proteins, Grd19p and Ypt35p, consist almost exclusively of the PX domain ( Table I). Assuming that signaling proteins are not likely to bind to a membrane for its own sake suggested that these PtdIns(3)P-specific PX domain-only proteins must also be involved in protein-protein interactions in order to transduce a signal from the membrane to some effector. This is further supported by several genetic observations. For example, yeast strains with a deletion of their GRD19 gene mis-sort the TGN membrane proteins A-ALP, Pep12p, and Kex2p to the vacuole (19,20).A direct involvement of the PX domain in protein-protein interactions has been observed in the p47 phox subunit of the neutrophil NADPH oxidase complex. p47 phox is held in an inactive state by an intramolecular interaction between its C-terminal Src homology 3 (SH3) domain and its PX domain. Upon activation by invading microorganisms, p47 phox becomes phosphorylated and the PX-SH3 interaction is disrupted (21, 22). The C-terminal SH3 domain interacts with a proline-rich region of the p47 phox -PX domain (13). Moreove...

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Section: Resultsmentioning

confidence: 61%

The Phox Homology (PX) Domain Protein Interaction Network in Yeast

Vollert¹,

Uetz²

2004

Molecular & Cellular Proteomics

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“…Size fractionation by gel filtration revealed that the amoebic retromerlike complex showed an apparent molecular weight of 300 -500 kDa (unpublished data), which is comparable to that in mammalian cells (Haft et al, 2000). The missing components, sorting nexins and Vps5/17, possess PX domain and BAR domain for the interaction with phosphatidyl inositol 3-phosphate and the curbed membrane (Seaman and Williams, 2002;Habermann, 2004;Seaman, 2005). We found proteins possessing either the BAR domain or the PX domain, but failed to find a protein containing both domains in the genome database.…”

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confidence: 87%

A Retromerlike Complex Is a Novel Rab7 Effector That Is Involved in the Transport of the Virulence Factor Cysteine Protease in the Enteric Protozoan ParasiteEntamoeba histolytica

Nakada‐Tsukui

Saito‐Nakano

Ali

et al. 2005

MBoC

139

173

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Vesicular trafficking plays an important role in a virulence mechanism of the enteric protozoan parasite Entamoeba histolytica as secreted and lysosomal cysteine protease (CP) contributes to both cytolysis of tissues and degradation of internalized host cells. Despite the primary importance of intracellular sorting in pathogenesis, the molecular mechanism of CP trafficking remains largely unknown. In this report we demonstrate that transport of CP is regulated through a specific interaction of Rab7A small GTPase (EhRab7A) with the retromerlike complex. The amoebic retromerlike complex composed of Vps26, Vps29, and Vps35 was identified as EhRab7A-binding proteins. The amoebic retromerlike complex specifically bound to GTP-EhRab7A, but not GDP-EhRab7A through the direct binding via the carboxy terminus of EhVps26. In erythrophagocytosis the retromerlike complex was recruited to prephagosomal vacuoles, the unique preparatory vacuole of digestive enzymes, and later to phagosomes. This dynamism was indistinguishable from that of EhRab7A, and consistent with the premise that the retromerlike complex is involved in the retrograde transport of putative hydrolase receptor(s) from preparatory vacuoles and phagosomes to the Golgi apparatus. EhRab7A overexpression caused enlargement of lysosomes and decrease of the cellular CP activity. The reduced CP activity was restored by the coexpression of EhVps26, implying that the EhRab7A-mediated transport of CP to phagosomes is regulated by the retromerlike complex. INTRODUCTIONRab GTPases play an essential role to regulate intracellular membrane trafficking. The compartmentalization and functions of Rab proteins are modulated at multiple layers of mechanisms including isoprenylation of the carboxy terminus, nucleotide exchange, and binding of specific effector molecules (Stenmark and Olkkonen, 2001;Takai et al., 2001;Tuvim et al., 2001). Among these modulators, effectors play key roles in the control of 7-90 Rab proteins depending on organisms from fission yeast and amoeba to mammals and plants. A variety of effectors have been identified and shown to interact with specific Rab protein. For instance, regulatory secretions of neurotransmitters from neurons or insulin from pancreatic ␤-cells are regulated by the specific interaction of Rab3 with its effectors (Jahn and Sudhof, 1999). At least four proteins, Rabphilin3, Rim1, Rim2, and Noc2, are known to bind Rab3 and recruit cAMP-GEFII, 14 -3-3, and zyxin, respectively, to regulate cAMP responsiveness, phosphoserine-dependent signaling, and the interaction with cytoskeleton (Kotake et al., 1997;Ozaki et al., 2000;Sun et al., 2003). The specificity of the Rab-effector interaction is attributable to primary sequence motifs in only a few cases including exophilins or Slip/Slac2, Rab3, and Rab27 effectors (Izumi et al., 2003). However, the majority of Rab effectors lack recognizable conserved binding motifs. For instance, Rab5 effectors, rabaptin-5, Rabex-5, EEA1, Rabenosyn-5, hVps34/p150, p110␤/p35␣, rabip4Ј, and rabankyrin-5, which a...

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“…The ability to grow in the absence of histidine (-His) is indicative of interactions. A summary of the results from three independent experiments is presented in Table 1 (a) Lysates from HeLa cells that were either not transfected (lanes 1 and 8, NT) or transfected with cDNAs encoding wild-type (lanes 2 and 9), IM-235,236-DD (lanes 3 and 10), Δ238-246 GG (lanes 4 and 11), 238-246 polyS (lanes 5 and 12), G238P (lanes 6 and 13) and R69A E71A (lanes 7 and 14) forms of myc-tagged Vps26 were subjected to immunoprecipitation (IP) using a mouse monoclonal antibody to the myc epitope The lysates (1% of the total, lanes 1-7) and immunoprecipitates (lanes [8][9][10][11][12][13][14] were subsequently analyzed by SDS-PAGE and immunoblotting (IB) with antibodies to the myc epitope (top panel), Vps35 (middle panel) or Vps29 (bottom panel). (b) The intracellular localization of Vps26-myc and the Vps26-myc mutants indicated in the figure (left column shows Alexa 488 green channel), as well as the colocalization of these constructs with SNX1 (middle column shows Alexa 546, red channel), was examined in fixed/permeabilized cells by indirect immunofluorescence and confocal microscopy.…”

Section: Supplementary Materialsmentioning

confidence: 99%

The retromer subunit Vps26 has an arrestin fold and binds Vps35 through its C-terminal domain

Shi

Rojas

Bonifacino

et al. 2006

Nat Struct Mol Biol

158

167

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The mammalian retromer complex consists of SNX1, SNX2, Vps26, Vps29, and Vps35, and retrieves lysosomal enzyme receptors from endosomes to the trans-Golgi network. The structure of human Vps26A at 2.1Å resolution reveals two curvedβ -sandwich domains connected by a polar core and a flexible linker. Vps26 has an unexpected structural relationship to arrestins. The Vps35-binding site on Vps26 maps to a mobile loop spanning residues 235-246, near the tip of the C-terminal domain. The loop is phylogenetically conserved and provides a mechanism for Vps26 integration into the complex that leaves the rest of the structure free for engagements with membranes and for conformational changes. Hydrophobic residues and a Gly in this loop are required for integration into the retromer complex and endosomal localization of human Vps26, and for the function of yeast Vps26 in carboxypeptidase Y sorting.The biosynthetic sorting of acid hydrolase precursors from the trans-Golgi network (TGN) to the endosomal-lysosomal system is central to the biogenesis of lysosomes in metazoans. In mammals, this sorting is directed by binding of mannose 6-phosphate groups on the hydrolases to two transmembrane receptors known as the cation-dependent and cation-independent mannose 6-phosphate receptors (CD-MPR and CI-MPR, respectively) 1 . At the TGN, the hydrolase-receptor complexes are packaged into carrier vesicles by several coat and adaptor proteins, including clathrin, AP-1 (adaptor protein 1) and GGA (Golgi-localized, gamma earcontaining, ADP ribosylation factor-binding) proteins 1,2 . These carrier vesicles bud from the TGN and fuse with endosomes. Within the endosome, the acid pH in the lumen triggers the release of the hydrolases from their receptors. The hydrolases are carried with the fluid phase to lysosomes, while the receptors return to the TGN to be reutilized in further rounds of sorting. Several proteins and complexes, including AP-1 3,4 , TIP47 (tail-interacting protein of 47 kDa) 5 and PACS-1 (phosphofurin acidic cluster sorting protein) 6 have been implicated in the transport of MPRs from endosomes to the TGN.A similar process has been described in yeast cells for the sorting of acid hydrolases to the vacuole, which is the fungal equivalent of the mammalian lysosome. Carboxypeptidase Y (CPY) is sorted by the Vps10 transmembrane receptor. Genetic screens in yeast have identified more than 60 Vps (vacuolar protein sorting) gene products 7 , which are involved in the transport of CPY to the vacuole. Five yeast Vps proteins, Vps5, Vps17, Vps26, Vps29 and Vps35, form a complex named "retromer" that is required for Vps10 sorting from endosomes to the late-5 To whom correspondence should be addressed: James H. Hurley, (301) 402-4703, fax (301) A homologous retromer complex consisting of five subunits termed SNX1, SNX2, Vps26, Vps29 and Vps35 has been described in humans 8 . Depletion of some of these subunits in vivo by RNA interference (RNAi) impairs retrieval of the CI-MPR and results in its missorting to lysosomes, where...

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Identification of the Functional Domains of Yeast Sorting Nexins Vps5p and Vps17p

Cited by 99 publications

References 32 publications

The Phox Homology (PX) Domain Protein Interaction Network in Yeast

The Phox Homology (PX) Domain Protein Interaction Network in Yeast

A Retromerlike Complex Is a Novel Rab7 Effector That Is Involved in the Transport of the Virulence Factor Cysteine Protease in the Enteric Protozoan ParasiteEntamoeba histolytica

The retromer subunit Vps26 has an arrestin fold and binds Vps35 through its C-terminal domain

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