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Query: EC:3.2.1.26 (invertase)
 4,927 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Carboxypeptidase Y from Saccharomyces cerevisiae contains 14% mannose, the only neutral sugar present. An antiserum can be raised in rabbits which reacts with both the protein and the sugar moieties of the enzyme. This antiserum also precipitates yeast invertase and yeast cell wall mannan. Thus carboxypeptidase Y, which is known to be localized in yeast vacuoles, is very probably a mannoprotein. Tunicamycin inhibits the apparent formation of carboxypeptidase Y to a similar extent as that of the externally localized mannoprotein, invertase. No accumulation of an inactive nonglycosylated or partly glycosylated carboxypeptidase Y occurs as determined by the immunoprecipitation technique. Tunicamycin also inhibits the apparent formation of proteinase A, whereas it does not affect the increase in the activities of a number of other enzymes. It is suggested that in the synthesis of glycoproteins there exists a regulatory link between the synthesis of their polypeptide chains and the reactions involved in their glycosylation.
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PMID:Inhibition of the apparent rate of synthesis on the vacuolar glycoprotein carboxypeptidase Y and its protein antigen by turicamycin in Saccharomyces cerevisiae. 79 Oct 99

The vacuolar ATPase of the yeast Saccharomyces cerevisiae acidifies the vacuolar lumen and generates an electrochemical gradient across the vacuole membrane. We have investigated the role of compartment acidification of the vacuolar system in the sorting of vacuolar proteins. Strains with chromosomal disruptions of genes (delta vat) encoding the A (69 x 10(3) M(r)), B (57 x 10(3) M(r)) or c (16 x 10(3) M(r)) subunits of the vacuolar ATPase accumulate and secrete precursor forms of the soluble vacuolar hydrolases carboxypeptidase Y and proteinase A. A kinetic analysis suggests that these precursor proteins accumulate in, and are secreted from, the Golgi complex or post-Golgi vesicles. In addition, subcellular fractionation shows that vacuolar hydrolase-invertase hybrid proteins are inefficiently localized to the vacuole in delta vat strains. This result suggests that the vat mutations cause a steady-state defect in vacuolar protein sorting. The vat mutations also affect the sorting of vacuolar membrane proteins. Precursor forms of alkaline phosphatase are accumulated in vat mutant cells, but to a lesser extent than is seen for the soluble vacuolar hydrolases. This finding, coupled with the insensitivity of alkaline phosphatase to the ATPase inhibitor bafilomycin A1, suggests that vacuolar membrane protein sorting is less sensitive to changes in lumenal pH when compared with the targeting of soluble vacuolar proteins. These results indicate that acidification of the vacuolar system is important for efficient sorting of soluble proteins to the vacuole.
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PMID:Mutations in the yeast vacuolar ATPase result in the mislocalization of vacuolar proteins. 149 Dec 35

We have isolated four yeast mutants that are unable to partition maternal vacuoles into growing buds. Three of these vacuole segregation (vac) mutants also mislocalize the vacuolar protease carboxypeptidase Y (CPY) to the cell surface, a phenotype previously reported for vac strains. A fourth mutant, vac2-1, exhibits a temperature-sensitive defect in vacuole segregation but does not show a defect in protein targeting from the Golgi apparatus to the vacuole. Haploid vac2-1 cells grown at the non-permissive temperature do not secrete CPY or a second vacuolar protease, proteinase A (PrA). Furthermore, newly synthesized precursors of CPY are converted to mature forms with similar kinetics in both vac2-1 and wild-type cells. In addition, invertase is secreted normally from vac2-1 cells, indicating that post-Golgi steps in the secretory pathway are not blocked in this mutant. These results suggest that VAC2 function is necessary for vacuole division and segregation in yeast but is not involved in vacuole protein sorting events at the Golgi apparatus.
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PMID:vac2: a yeast mutant which distinguishes vacuole segregation from Golgi-to-vacuole protein targeting. 205 Jan 11

We have investigated the vacuolar delivery of alpha-mannosidase, a marker enzyme of the vacuolar membrane in the yeast Saccharomyces cerevisiae, and found that the enzyme has several unique characteristics in its biosynthesis and vacuolar delivery. alpha-Mannosidase has no typical signal sequence (Yoshihisa, T., and Anraku, Y. (1989) Biochem. Biophys. Res. Commun. 163, 908-915) but is located on the inner surface of the vacuolar membrane. The enzyme is synthesized as a 107-kDa polypeptide and converted to a 73-kDa polypeptide. Although the conversion depends on a vacuolar processing protease, proteinase A, it is much slower (t1/2 = 10 h) than the proteinase A-dependent processing of other vacuolar proteins. None of Asn-X-Thr/Ser sites on the 107-kDa alpha-mannosidase or on two alpha-mannosidase-invertase fusion proteins that are localized inside the vacuole receives N-linked oligosaccharide, whereas those sites on a carboxypeptidase Y-alpha-mannosidase fusion protein are N-glycosylated. The newly synthesized alpha-mannosidase is normally delivered to the vacuole and converted to the 73-kDa polypeptide even when the secretory pathway is blocked by a subset of sec mutations. These characteristics are different from those of other vacuolar proteins targeted to the vacuole via the secretory pathway. We conclude that alpha-mannosidase is delivered to the vacuole in a novel pathway separate from the secretory pathway.
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PMID:A novel pathway of import of alpha-mannosidase, a marker enzyme of vacuolar membrane, in Saccharomyces cerevisiae. 226 33

Using a selection for spontaneous mutants that mislocalize a vacuolar carboxypeptidase Y (CPY)-invertase fusion protein to the cell surface, we identified vacuolar protein targeting (vpt) mutants in 25 new vpt complementation groups. Additional alleles in each of the eight previously identified vpt complementation groups (vpt1 through vpt8) were also obtained. Representative alleles from each of the 33 vpt complementation groups (vpt1 through vpt33) were shown to exhibit defects in the sorting and processing of several native vacuolar proteins, including the soluble hydrolases CPY, proteinase A, and proteinase B. Of the 33 complementation groups, 19 were found to contain mutant alleles that led to extreme defects. In these mutants, CPY accumulated in its Golgi complex-modified precursor form which was secreted by the mutant cells. Normal protein secretion appeared to be unaffected in the vpt mutants. The lack of significant leakage of cytosolic markers from the vpt mutant cells indicated that the vacuolar protein-sorting defects associated with these mutants do not result from cell lysis. In addition, the observation that the precursor rather than the mature forms of CPY, proteinase A, proteinase B were secreted from the vpt mutants was consistent with the fact that mislocalization occurred at a stage after Golgi complex-specific modification, but before final vacuolar sorting of these enzymes. Vacuolar membrane protein sorting appeared to be unaffected in the majority of the vpt mutants. However, a subset of the vpt mutants (vpt11, vpt16, vpt18, and vpt33) was found to exhibit defects in the sorting of a vacuolar membrane marker enzyme, alpha-mannosidase. Up to 50% of the alpha-mannosidase enzyme activity was found to be mislocalized to the cell surface in these vpt mutants. Seven of the vpt complementation groups (vpt3, vpt11, vpt15, vpt16, vpt18, vpt29, and vpt33) contained alleles that led to a conditional lethal phenotype; the mutants were temperature sensitive for vegetative cell growth. This temperature-sensitive phenotype has been shown to be recessive and to cosegregate with the vacuolar protein-sorting defect in each case. Tetrad analysis showed that vpt3 mapped to the right arm of chromosome XV and that vpt15 mapped to the right arm of chromosome II. Intercrosses with other mutants that exhibited defects in vacuolar protein sorting or function (vpl, sec, pep, and end mutants) revealed several overlaps among these different sets of genes. Together, these data indicate that more than 50 gene products are involved, directly or indirectly, in the process of vacuolar protein sorting.
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PMID:Protein sorting in Saccharomyces cerevisiae: isolation of mutants defective in the delivery and processing of multiple vacuolar hydrolases. 306 74

An inactive precursor form of proteinase A (PrA) transits through the early secretory pathway before final vacuolar delivery. We used gene fusions between the gene coding for PrA (PEP4) and the gene coding for the secretory enzyme invertase (SUC2) to identify vacuolar protein-sorting information in the PrA precursor. We found that the 76-amino-acid preprosegment of PrA contains at least two sorting signals: an amino-terminal signal peptide that is cleaved from the protein at the level of the endoplasmic reticulum followed by the prosegment which functions as a vacuolar protein-sorting signal. PrA-invertase hybrid proteins that carried this sequence information were accurately sorted to the yeast vacuole as determined by cell fractionation and immunolocalization studies. Hybrid proteins lacking all or a portion of the PrA prosegment were secreted from the cell. Our gene fusion data together with an analysis of the wild-type PrA protein indicated that N-linked carbohydrate modifications are not required for vacuolar sorting of this protein. Furthermore, results obtained with a set of deletion mutations constructed in the PrA prosegment indicated that this sequence also contributes to proper folding of this polypeptide into a stable transit-competent molecule.
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PMID:Intracellular sorting and processing of a yeast vacuolar hydrolase: proteinase A propeptide contains vacuolar targeting information. 329 Jun 49

The vacuole of the yeast Saccharomyces cerevisiae contains a proton-translocating ATPase that acidifies the vacuolar lumen and generates an electrochemical potential across the vacuole membrane. Strains with chromosomal disruptions of the genes encoding the A, B, and c subunits of the vacuolar ATPase accumulate precursor forms of the vacuolar membrane protein alkaline phosphatase, and the soluble vacuolar hydrolases carboxypeptidase Y and proteinase A. We have found that the intracellular precursors in delta vat strains accumulate within the secretory pathway at some point before delivery to the vacuole but after transit to the Golgi complex. Purified vacuoles from delta vat cells do not contain the precursor forms of carboxypeptidase Y or alkaline phosphatase. In addition, vacuolar hydrolase-invertase hybrid proteins are inefficiently delivered to the vacuole in delta vat strains as demonstrated by vacuole isolation. Further subcellular fractionation to separate organelles indicate that significant amounts of the carboxypeptidase Y-invertase and alkaline phosphatase-invertase hybrid proteins are located in the late Golgi complex and/or post Golgi compartments.
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PMID:Vacuolar ATPase mutants accumulate precursor proteins in a pre-vacuolar compartment. 848 10

The sorting of the yeast proteases proteinase A and carboxypeptidase Y to the vacuole is a saturable, receptor-mediated process. Information sufficient for vacuolar sorting of the normally secreted protein invertase has in fusion constructs previously been found to reside in the propeptide of proteinase A. We found that sorting of such a hybrid protein is dependent on the vacuolar protein-sorting receptor Vps10p. This was unexpected, as strains disrupted for VPS10 sort more than 85% of the proteinase A to the vacuole. Consistent with a role for Vps10p in sorting of proteinase A, we found that 1) overproduction of Vps10p suppressed the missorting phenotype associated with overproduction of proteinase A, 2) overproduction of proteinase A induced missorting of carboxypeptidase Y, 3) vacuolar sorting of proteinase A in a deltavps10 strain was readily saturated by modest overproduction of proteinase A, and 4) Vps10p and proteinase A interact directly and specifically as shown by chemical cross-linking. Interestingly, overexpression of two telomere-linked VPS10 homologues, VTH1 and VTH2 suppressed the missorting phenotypes of a deltavps10 strain. However, disruption of the VTH1 and VTH2 genes did not affect the sorting of proteinase A. We conclude that proteinase A utilizes at least two mechanisms for sorting, a Vps10p-dependent path and a Vth1p/Vth2p/Vps10p-independent path.
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PMID:Multiple pathways for vacuolar sorting of yeast proteinase A. 866 42

Vp10p is a receptor that sorts several different vacuolar proteins by cycling between a late Golgi compartment and the endosome. The cytoplasmic tail of Vps10p is necessary for the recycling, whereas the lumenal domain is predicted to interact with the soluble ligands. We have studied ligand binding to Vps10p by introducing deletions in the lumenal region. This region contains two domains with homology to each other. Domain 2 binds carboxypeptidase Y (CPY), proteinase A (PrA) and hybrids of these proteases with invertase. Moreover, we show that aminopeptidase Y (APY) is a ligand of Vps10p. The native proteases compete for binding to domain 2. Binding of CPY(156)-invertase or PrA(137)-invertase, on the other hand, do not interfere with binding of CPY to Vps10p. Furthermore, the Q24RPL27 sequence known to be important for vacuolar sorting of CPY, is of little importance in the Vps10p-dependent sorting of CPY-invertase. Apparently, domain 2 contains two different binding sites; one for APY, CPY and PrA, and one for CPY-invertase and PrA-invertase. The latter interaction seems not to be sequence specific, and we suggest that an unfolded structure in these ligands is recognized by Vps10p.
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PMID:Ligand recognition and domain structure of Vps10p, a vacuolar protein sorting receptor in Saccharomyces cerevisiae. 1009 82