Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
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The proper localization of resident membrane proteins to the trans-Golgi network (TGN) involves mechanisms for both TGN retention and retrieval from post-TGN compartments. In this study we report identification of a new gene, GRD20, involved in protein sorting in the TGN/endosomal system of Saccharomyces cerevisiae. A strain carrying a transposon insertion allele of GRD20 exhibited rapid vacuolar degradation of the resident TGN endoprotease Kex2p and aberrantly secreted approximately 50% of the soluble vacuolar hydrolase carboxypeptidase Y. The Kex2p mislocalization and carboxypeptidase Y missorting phenotypes were exhibited rapidly after loss of Grd20p function in grd20 temperature-sensitive mutant strains, indicating that Grd20p plays a direct role in these processes. Surprisingly, little if any vacuolar degradation was observed for the TGN membrane proteins A-ALP and Vps10p, underscoring a difference in trafficking patterns for these proteins compared with that of Kex2p. A grd20 null mutant strain exhibited extremely slow growth and a defect in polarization of the actin cytoskeleton, and these two phenotypes were invariably linked in a collection of randomly mutagenized grd20 alleles. GRD20 encodes a hydrophilic protein that partially associates with the TGN. The discovery of GRD20 suggests a link between the cytoskeleton and function of the yeast TGN.
Mol Biol Cell 1999 Dec
PMID:The yeast GRD20 gene is required for protein sorting in the trans-Golgi network/endosomal system and for polarization of the actin cytoskeleton. 1058 57

The late Golgi of the yeast Saccharomyces cerevisiae receives membrane traffic from the secretory pathway as well as retrograde traffic from post-Golgi compartments, but the machinery that regulates these vesicle-docking and fusion events has not been characterized. We have identified three components of a novel protein complex that is required for protein sorting at the yeast late Golgi compartment. Mutation of VPS52, VPS53, or VPS54 results in the missorting of 70% of the vacuolar hydrolase carboxypeptidase Y as well as the mislocalization of late Golgi membrane proteins to the vacuole, whereas protein traffic through the early part of the Golgi complex is unaffected. A vps52/53/54 triple mutant strain is phenotypically indistinguishable from each of the single mutants, consistent with the model that all three are required for a common step in membrane transport. Native coimmunoprecipitation experiments indicate that Vps52p, Vps53p, and Vps54p are associated in a 1:1:1 complex that sediments as a single peak on sucrose velocity gradients. This complex, which exists both in a soluble pool and as a peripheral component of a membrane fraction, colocalizes with markers of the yeast late Golgi by immunofluorescence microscopy. Together, the phenotypic and biochemical data suggest that VPS52, VPS53, and VPS54 are required for the retrograde transport of Golgi membrane proteins from an endosomal/prevacuolar compartment. The Vps52/53/54 complex joins a growing list of distinct multisubunit complexes that regulate membrane-trafficking events.
Mol Biol Cell 2000 Jan
PMID:Vps52p, Vps53p, and Vps54p form a novel multisubunit complex required for protein sorting at the yeast late Golgi. 1063 10

The Sec23p/Sec24p complex functions as a component of the COPII coat in vesicle transport from the endoplasmic reticulum. Here we characterize Saccharomyces cerevisiae SEC24, which encodes a protein of 926 amino acids (YIL109C), and a close homologue, ISS1 (YNL049C), which is 55% identical to SEC24. SEC24 is essential for vesicular transport in vivo because depletion of Sec24p is lethal, causing exaggeration of the endoplasmic reticulum and a block in the maturation of carboxypeptidase Y. Overproduction of Sec24p suppressed the temperature sensitivity of sec23-2, and overproduction of both Sec24p and Sec23p suppressed the temperature sensitivity of sec16-2. SEC24 gene disruption could be complemented by overexpression of ISS1, indicating functional redundancy between the two homologous proteins. Deletion of ISS1 had no significant effect on growth or secretion; however, iss1Delta mutants were found to be synthetically lethal with mutations in the v-SNARE genes SEC22 and BET1. Moreover, overexpression of ISS1 could suppress mutations in SEC22. These genetic interactions suggest that Iss1p may be specialized for the packaging or the function of COPII v-SNAREs. Iss1p tagged with His(6) at its C terminus copurified with Sec23p. Pure Sec23p/Iss1p could replace Sec23p/Sec24p in the packaging of a soluble cargo molecule (alpha-factor) and v-SNAREs (Sec22p and Bet1p) into COPII vesicles. Abundant proteins in the purified vesicles produced with Sec23p/Iss1p were indistinguishable from those in the regular COPII vesicles produced with Sec23p/Sec24p.
Mol Biol Cell 2000 Mar
PMID:Sec24p and Iss1p function interchangeably in transport vesicle formation from the endoplasmic reticulum in Saccharomyces cerevisiae. 1071 14

Sialidosis is an autosomal recessive disease caused by the genetic deficiency of lysosomal sialidase, which catalyzes the hydrolysis of sialoglycoconjugates. The disease is associated with progressive impaired vision, macular cherry-red spots and myoclonus (sialidosis type I) or with skeletal dysplasia, Hurler-like phenotype, dysostosis multiplex, mental retardation and hepatosplenomegaly (sialidosis type II). We have analyzed the genomic DNA from nine sialidosis patients of multiple ethnic origin in order to find mutations responsible for the enzyme deficiency. The activity of the identified variants was studied by transgenic expression. One patient had a frameshift mutation (G623delG deletion), which introduced a stop codon, truncating 113 amino acids. All others had missense mutations: G679G-->A (Gly227Arg), C893C-->T (Ala298Val), G203G-->T (Gly68Val), A544A-->G (Ser182Gly) C808C-->T (Leu270Phe) and G982G-->A (Gly328Ser). We have modeled the three-dimensional structure of sialidase based on the atomic coordinates of the homologous bacterial sialidases, located the positions of mutations and estimated their potential effect. This analysis showed that five mutations are clustered in one region on the surface of the sialidase molecule. These mutations dramatically reduce the enzyme activity and cause a rapid intralysosomal degradation of the expressed protein. We hypothesize that this region may be involved in the interface of sialidase binding with lysosomal cathepsin A and/or beta-galactosidase in their high-molecular-weight complex required for the expression of sialidase activity in the lysosome.
Hum Mol Genet 2000 Apr 12
PMID:Characterization of the sialidase molecular defects in sialidosis patients suggests the structural organization of the lysosomal multienzyme complex. 1076 32

We have characterized LUV1/RKI1/TCS3/VPS54, a novel yeast gene required to maintain normal vacuolar morphology. The luv1 mutant was identified in a genetic screen for mutants requiring the phosphatase calcineurin for vegetative growth. luv1 mutants lack a morphologically intact vacuole and instead accumulate small vesicles that are acidified and contain the vacuolar proteins alkaline phosphatase and carboxypeptidase Y and the vacuolar membrane H(+)-ATPase. Endocytosis appears qualitatively normal in luv1 mutants, but some portion (28%) of carboxypeptidase Y is secreted. luv1 mutants are sensitive to several ions (Zn(2+), Mn(2+), and Cd(2+)) and to pH extremes. These mutants are also sensitive to hygromycin B, caffeine, and FK506, a specific inhibitor of calcineurin. Some vacuolar protein-sorting mutants display similar drug and ion sensitivities, including sensitivity to FK506. Luv1p sediments at 100,000 x g and can be solubilized by salt or carbonate, indicating that it is a peripheral membrane protein. A Green Fluorescent Protein-Luv1 fusion protein colocalizes with the dye FM 4-64 at the endosome, and hemagglutinin-tagged Luv1p colocalizes with the trans-Golgi network/endosomal protease Kex2p. Computer analysis predicts a short coiled-coil domain in Luv1p. We propose that this protein maintains traffic through or the integrity of the early endosome and that this function is required for proper vacuolar morphology.
Mol Biol Cell 2000 Jul
PMID:Luv1p/Rki1p/Tcs3p/Vps54p, a yeast protein that localizes to the late Golgi and early endosome, is required for normal vacuolar morphology. 1088 79

We show that the vacuolar protein sorting gene VPS44 is identical to NHX1, a gene that encodes a sodium/proton exchanger. The Saccharomyces cerevisiae protein Nhx1p shows high homology to mammalian sodium/proton exchangers of the NHE family. Nhx1p is thought to transport sodium ions into the prevacuole compartment in exchange for protons. Pulse-chase experiments show that approximately 35% of the newly synthesized soluble vacuolar protein carboxypeptidase Y is missorted in nhx1 delta cells, and is secreted from the cell. nhx1 delta cells accumulate late Golgi, prevacuole, and lysosome markers in an aberrant structure next to the vacuole, and late Golgi proteins are proteolytically cleaved more rapidly than in wild-type cells. Our results show that efficient transport out of the prevacuolar compartment requires Nhx1p, and that nhx1 delta cells exhibit phenotypes characteristic of the "class E" group of vps mutants. In addition, we show that Nhx1p is required for protein trafficking even in the absence of the vacuolar ATPase. Our analysis of Nhx1p provides the first evidence that a sodium/proton exchange protein is important for correct protein sorting, and that intraorganellar ion balance may be important for endosomal function in yeast.
Mol Biol Cell 2000 Dec
PMID:The sodium/proton exchanger Nhx1p is required for endosomal protein trafficking in the yeast Saccharomyces cerevisiae. 1110 23

Soybean glycinin is a member of the 11 S globulin family. The crystal structure of proglycinin was determined by X-ray crystallography at 2.8 A resolution with an R-factor of 0.199 and a free R-factor of 0.250. A trimer molecule was found in an asymmetric unit of crystals. The trimer model contains three A1aB1b subunits and comprises 1128 amino acid residues and 34 water molecules. The constituent protomers of the homo-trimeric protein are arranged around a 3-fold symmetry axis with dimensions of 95 Ax95 Ax40 A. The protomer model is composed of five fragments which correspond roughly to conserved regions based on the sequence alignment of various 11 S globulins. The core of the protomer consists of two jelly-roll beta-barrels and two extended helix domains. This structure of proglycinin is similar to those of canavalin and phaseolin belonging to the 7 S globulin family, strongly supporting the hypothesis that both 7 S and 11 S globulins are derived from a common ancestor. The inter and intra-chain disulfide bonds conserved in the 11 S globulin family are clearly observed. It is found that the face with the inter-chain disulfide bond (IE face) contains more hydrophobic residues than that with the intra-chain disulfide bond. This suggests that a mature hexamer is formed by the interaction between the IE faces after processing.
J Mol Biol 2001 Jan 12
PMID:Crystal structure of soybean proglycinin A1aB1b homotrimer. 1112 7

A native immunoisolation procedure has been used to investigate the role of clathrin-coated vesicles (CCVs) in the transport of vacuolar proteins between the trans-Golgi network (TGN) and the prevacuolar/endosome compartments in the yeast Saccharomyces cerevisiae. We find that Apl2p, one large subunit of the adaptor protein-1 complex, and Vps10p, the carboxypeptidase Y vacuolar protein receptor, are associated with clathrin molecules. Vps10p packaging in CCVs is reduced in pep12 Delta and vps34 Delta, two mutants that block Vps10p transport from the TGN to the endosome. However, Vps10p sorting is independent of Apl2p. Interestingly, a Vps10C(t) Delta p mutant lacking its C-terminal cytoplasmic domain, the portion of the receptor responsible for carboxypeptidase Y sorting, is also coimmunoprecipitated with clathrin. Our results suggest that CCVs mediate Vps10p transport from the TGN to the endosome independent of direct interactions between Vps10p and clathrin coats. The Vps10p C-terminal domain appears to play a principal role in retrieval of Vps10p from the prevacuolar compartment rather than in sorting from the TGN.
Mol Biol Cell 2001 Feb
PMID:Vps10p transport from the trans-Golgi network to the endosome is mediated by clathrin-coated vesicles. 1117 29

Cathepsin A/protective protein [3.4.16.5], carboxypeptidase A, is a lysosomal serine protease with structural homology to yeast (Saccharomyces cerevisiae) carboxypeptidase Y. Cathepsin A is a member of the alpha/beta hydrolase fold family and has been suggested to share a common ancestral relationship with other alpha/beta hydrolase fold enzymes, such as cholinesterases. Several lines of evidence indicate that cathepsin A is a multicatalytic enzyme with deamidase and esterase in addition to carboxypeptidase activities. Cathepsin A was recently identified in human platelets as deamidase. In vitro, it hydrolyzes a variety of bioactive peptide hormones including tachykinins, suggesting that extralysosomal cathepsin A plays a role in regulation of bioactive peptide functions. Recent reports emphasize the lysosomal protective function of cathepsin A rather than its protease function. The protective function of cathepsin A is distinct from its catalytic function. Human lysosomal beta-galactosidase and neuraminidase exist as a high molecular weight enzyme complex, in which there is a 54-kDa glycoprotein termed 'lysosomal protective protein'. Based on cell culture studies, protective protein was found to protect both beta-galactosidase and neuraminidase from intralysosomal proteolysis by forming a multienzyme complex and was shown to be deficient in patients with galactosialidosis, a combined deficiency of beta-galactosidase and neuraminidase. Molecular cloning and gene expression studies have disclosed that protective protein is cathepsin A. The cathepsin A precursor has the potential to restore both beta-galactosidase and neuraminidase activities in fibroblasts from patients with galactosialidosis. Cathepsin A knockout mice showed a phenotype similar to human galactosialidosis and the deficient phenotype found in the mutant mice was corrected by transplanting erythroid precursor cells overexpressing cathepsin A. Collectively, these findings demonstrate the significance of cathepsin A as a key molecule in the onset of galactosialidosis and also highlight the therapeutic potential of the cathepsin A precursor for patients with galactosialidosis.
Cell Mol Life Sci 1999 Dec
PMID:Cathepsin A/protective protein: an unusual lysosomal multifunctional protein. 1121 24

The multispanning membrane protein Ste6, a member of the ABC-transporter family, is transported to the yeast vacuole for degradation. To identify functions involved in the intracellular trafficking of polytopic membrane proteins, we looked for functions that block Ste6 transport to the vacuole upon overproduction. In our screen, we identified several known vacuolar protein sorting (VPS) genes (SNF7/VPS32, VPS4, and VPS35) and a previously uncharacterized open reading frame, which we named MOS10 (more of Ste6). Sequence analysis showed that Mos10 is a member of a small family of coiled-coil-forming proteins, which includes Snf7 and Vps20. Deletion mutants of all three genes stabilize Ste6 and show a "class E vps phenotype." Maturation of the vacuolar hydrolase carboxypeptidase Y was affected in the mutants and the endocytic tracer FM4-64 and Ste6 accumulated in a dot or ring-like structure next to the vacuole. Differential centrifugation experiments demonstrated that about half of the hydrophilic proteins Mos10 and Vps20 was membrane associated. The intracellular distribution was further analyzed for Mos10. On sucrose gradients, membrane-associated Mos10 cofractionated with the endosomal t-SNARE Pep12, pointing to an endosomal localization of Mos10. The growth phenotypes of the mutants suggest that the "Snf7-family" members are involved in a cargo-specific event.
Mol Biol Cell 2001 Mar
PMID:A family of small coiled-coil-forming proteins functioning at the late endosome in yeast. 1125 Oct 82


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