Horazdovsky, B. F. et al. A sorting nexin-1 homologue, Vps5p, forms a complex with Vps17p and is required for recycling the vacuolar protein-sorting receptor. Mol. Biol. Cell 8, 1529-1541

Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas 75235-9038, USA.
Molecular Biology of the Cell (Impact Factor: 4.47). 09/1997; 8(8):1529-41. DOI: 10.1091/mbc.8.8.1529
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A number of the Saccharomyces cerevisiae vacuolar protein-sorting (vps) mutants exhibit an altered vacuolar morphology. Unlike wild-type cells that contain 1-3 large vacuolar structures, the class B vps5 and vps17 mutant cells contain 10-20 smaller vacuole-like compartments. To explore the role of these VPS gene products in vacuole biogenesis, we cloned and sequenced VPS5 and characterized its protein products. The VPS5 gene is predicted to encode a very hydrophilic protein of 675 amino acids that shows significant sequence homology with mammalian sorting nexin-1. Polyclonal antiserum directed against the VPS5 gene product detects a single, cytoplasmic protein that is phosphorylated specifically on a serine residue(s). Subcellular fractionation studies indicate that Vps5p is associated peripherally with a dense membrane fraction distinct from Golgi, endosomal, and vacuolar membranes. This association was found to be dependent on the presence of another class B VPS gene product, Vps17p. Biochemical cross-linking studies demonstrated that Vps5p and Vps17p physically interact. Gene disruption experiments show that the VPS5 genes product is not essential for cell viability; however, cells carrying the null allele contain fragmented vacuoles and exhibit defects in vacuolar protein-sorting similar to vps17 null mutants. More than 95% of carboxypeptidase Y is secreted from these cells in its Golgi-modified p2 precursor form. Additionally, the Vps10p vacuolar protein-sorting receptor is mislocalized to the vacuole in vps5 mutant cells. On the basis of these and other observations, we propose that the Vps17p protein complex may participate in the intracellular trafficking of the Vps10p-sorting receptor, as well as other later-Golgi proteins.

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Available from: Matthew Seaman, Feb 26, 2014
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    • "Some of the possible targets of PI3P during sporulation have been identified, including two sorting nexins, Vps5 and Vps17, and the FYVE domain–containing protein Sst4/Vps27. Vps5 and Vps17 have a phox homology (PX) domain, and analysis of homologues of these proteins in S. cerevisiae suggests that Vps5 and Vps17 in S. pombe act as PI3P-dependent mediators of retrograde trafficking from the endosome to the Golgi apparatus, which in turn acts as an efficient anterograde membrane flux to the FSM (Horazdovsky et al., 1997; Pfeffer, 2001; Onishi et al., 2003a; Koga et al., 2004). In contrast, Sst4 functions at later stages of FSM formation, specifically when the FSM closes (Onishi et al., 2007). "
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    ABSTRACT: Sporulation in fission yeast represents a unique mode of cell division in which a new cell is formed within the cytoplasm of a mother cell. This event is accompanied by formation of the forespore membrane (FSM), which becomes the plasma membrane of spores. At prophase II, the spindle pole body (SPB) forms an outer plaque, from which formation of the FSM is initiated. Several components of the SPB play an indispensable role in SPB modification, and therefore in sporulation. In this paper, we report the identification of a novel SPB component, Spo7, which has a pleckstrin homology (PH) domain. We found that Spo7 was essential for initiation of FSM assembly, but not for SPB modification. Spo7 directly bound to Meu14, a component of the leading edge of the FSM, and was essential for proper localization of Meu14. The PH domain of Spo7 had affinity for phosphatidylinositol 3-phosphate (PI3P). spo7 mutants lacking the PH domain showed aberrant spore morphology, similar to that of meu14 and phosphatidylinositol 3-kinase (pik3) mutants. Our study suggests that Spo7 coordinates formation of the leading edge and initiation of FSM assembly, thereby accomplishing accurate formation of the FSM.
    Molecular biology of the cell 07/2011; 22(18):3442-55. DOI:10.1091/mbc.E11-02-0125 · 4.47 Impact Factor
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    • "The presence of components of the large subunit (VPS26A, VPS29, and VPS35C) on the multivesicular body (MVB) has been demonstrated by immunoelectron microscopy (Oliviusson et al., 2006). Each lossof-function yeast mutant lacking a retromer component exhibits a similar phenotype, such as mis-sorting of the CPY out of the cell (Horazdovsky et al., 1997; Nothwehr and Hindes, 1997; Seaman et al., 1997; Nothwehr et al., 1999; Reddy and Seaman, 2001). Similarly, all three Arabidopsis mutants, T-DNA insertion mutants of VPS26A or VPS35A and mag1-1 with reduced expression of VPS29, partially suppressed the zig-1 phenotype (Figure 5). "
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    ABSTRACT: Arabidopsis thaliana zigzag (zig) is a loss-of-function mutant of Qb-SNARE VTI11, which is involved in membrane trafficking between the trans-Golgi network and the vacuole. zig-1 exhibits abnormalities in shoot gravitropism and morphology. Here, we report that loss-of-function mutants of the retromer large subunit partially suppress the zig-1 phenotype. Moreover, we demonstrate that three paralogous VPS35 genes of Arabidopsis have partially overlapping but distinct genetic functions with respect to zig-1 suppression. Tissue-specific complementation experiments using an endodermis-specific SCR promoter show that expression of VPS35B or VPS35C cannot complement the function of VPS35A. The data suggest the existence of functionally specialized paralogous VPS35 genes that nevertheless share common functions.
    The Plant Cell 03/2010; 22(1):159-72. DOI:10.1105/tpc.109.069294 · 9.34 Impact Factor
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    • "Vps26p has an arrestin fold, and the C-terminal tail of Vps26 interacts with Vps35p via its C-terminal domain (Shi et al., 2006; Collins et al., 2008). In yeast, the small subunit of retromer comprises the proteins Vps5p and Vps17p (Horazdovsky et al., 1997). These proteins have in common a phox homology (PX) domain that facilitates binding to phosphoinositides (Carlton et al., 2005), and also a BAR (Bin, amphiphysin, Rvs) domain that, through coiled-coil (CC) interactions, forms a curved dimer with a net positive charge on its concave side. "
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    ABSTRACT: Receptor-mediated sorting processes in the secretory pathway of eukaryotic cells rely on mechanisms to recycle the receptors after completion of transport. Based on this principle, plant vacuolar sorting receptors (VSRs) are thought to recycle after dissociating of receptor-ligand complexes in a pre-vacuolar compartment. This recycling is mediated by retromer, a cytosolic coat complex that comprises sorting nexins and a large heterotrimeric subunit. To analyse retromer-mediated VSR recycling, we have used a combination of immunoelectron and fluorescence microscopy to localize the retromer components sorting nexin 1 (SNX1) and sorting nexin 2a (SNX2a) and the vacuolar sorting protein VPS29p. All retromer components localize to the trans-Golgi network (TGN), which is considered to represent the early endosome of plants. In addition, we show that inhibition of retromer function in vivo by expression of SNX1 or SNX2a mutants as well as transient RNAi knockdown of all sorting nexins led to accumulation of the VSR BP80 at the TGN. Quantitative protein transport studies and live-cell imaging using fluorescent vacuolar cargo molecules revealed that arrival of these VSR ligands at the vacuole is not affected under these conditions. Based on these findings, we propose that the TGN is the location of retromer-mediated recycling of VSRs, and that transport towards the lytic vacuole downstream of the TGN is receptor-independent and occurs via maturation, similar to transition of the early endosome into the late endosome in mammalian cells.
    The Plant Journal 10/2009; 61(1):107-21. DOI:10.1111/j.1365-313X.2009.04034.x · 5.97 Impact Factor
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