Vacuolar and extracellular maturation of Saccharomyces cerevisiae proteinase A

Wolff, Anne Mette; Din, Nanni; Petersen, J.

doi:10.1002/(sici)1097-0061(199607)12:9<823::aid-yea975>3.0.co;2-j

Cited by 28 publications

(19 citation statements)

References 36 publications

(37 reference statements)

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“…However, it has been shown that PEP4 is secreted (instead of targeted to the vacuole) upon overexpression (34), and the protein has also been found in highly purified mitochondria, indicating that the vacuole may not be the only or predominant destination of PEP4. Four proteins (GRX7, SSA2, YKT6, and YKL077W) with shared localization in vacuoles and another organelle were depleted in our experiment.…”

Section: Proteins With Annotated Vacuolar Localizationmentioning

confidence: 99%

The Yeast Vacuolar Membrane Proteome

Wiederhold

Gandhi

Permentier

et al. 2009

Molecular & Cellular Proteomics

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Transport of solutes between the cytosol and the vacuolar lumen is of crucial importance for various functions of vacuoles, including ion homeostasis; detoxification; storage of different molecules such as amino acids, phosphate, and calcium ions; and proteolysis. To identify proteins that catalyze solute transport across the vacuolar membrane, the membrane proteome of purified Saccharomyces cerevisiae vacuoles was analyzed. Subtractive proteomics was used to distinguish contaminants from true vacuolar proteins by comparing the relative abundances of proteins in pure and crude preparations. A robust statistical analysis combining enrichment ranking with the double boundary iterative group analysis revealed that 148 proteins were significantly enriched in the pure vacuolar preparations. Among these proteins were well characterized vacuolar proteins, such as the subunits of the vacuolar H ؉ -ATPase, but also proteins that had not previously been assigned to a cellular location, many of which are likely novel vacuolar membrane transporters, e.g. for nucleosides and oligopeptides. Although the majority of contaminating proteins from other organelles were depleted from the pure vacuolar membranes, some proteins annotated to reside in other cellular locations were enriched along with the vacuolar proteins. In many cases the enrichment of these proteins is biologically relevant, and we discuss that a large group is involved in membrane fusion and protein trafficking to vacuoles and may have multiple localizations. Other proteins are degraded in vacuoles, and in some cases database annotations are likely to be incomplete or incorrect. Our work provides a wealth of information on vacuolar biology and a solid basis for further characterization of vacuolar functions. The pH difference of ϳ1.7 pH units between the vacuolar lumen and the cytosol is used as the driving force for substrate-proton antiport systems in the vacuolar membrane (1). Transport processes across the vacuolar membrane are important for many crucial functions of vacuoles: storage of organic molecules such as polyphosphates, mannans, and other carbohydrates (2, 3); detoxification, i.e. removal and accumulation of harmful substances such as heavy metals and drugs; and proton and ion homeostasis. Another major function of lysosomes and vacuoles is the intracellular proteolysis of cytosolic and membrane proteins (4, 5) and turnover of organelles, e.g. lipid bodies, mitochondria, peroxisomes, and portions of nuclei (6 -9). Hydrolytic proteins in the lumen of vacuoles, including proteases, lipases, phosphatases, and nucleases, carry out these functions. Whereas vacuolar luminal proteins have been studied fairly extensively (10, 11), our knowledge about the integral membrane proteins in the yeast vacuolar membrane is limited.

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Section: Proteins With Annotated Vacuolar Localizationmentioning

confidence: 99%

The Yeast Vacuolar Membrane Proteome

Wiederhold

Gandhi

Permentier

et al. 2009

Molecular & Cellular Proteomics

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“…Interestingly, maturation of most vacuolar hydrolases occurs, albeit at reduced levels, in strains in which subunits of the vacuolar ATPase had been deleted or in the presence of ATPase inhibitors. GPD1 degradation could require denaturation of GPD1 by the low vacuolar pH, turning the protein into a better substrate for the vacuolar proteases, whereas protease precursor processing does not depend on a low pH (Wolff et al, 1996).…”

Section: Figurementioning

confidence: 99%

Import into and Degradation of Cytosolic Proteins by Isolated Yeast Vacuoles

Horst

Knecht

Schu

1999

MBoC

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In eukaryotic cells, both lysosomal and nonlysosomal pathways are involved in degradation of cytosolic proteins. The physiological condition of the cell often determines the degradation pathway of a specific protein. In this article, we show that cytosolic proteins can be taken up and degraded by isolated Saccharomyces cerevisiae vacuoles. After starvation of the cells, protein uptake increases. Uptake and degradation are temperature dependent and show biphasic kinetics. Vacuolar protein import is dependent on cytosolic heat shock proteins of the hsp70 family and on protease-sensitive component(s) on the outer surface of vacuoles. Degradation of the imported cytosolic proteins depends on a functional vacuolar ATPase. We show that the cytosolic isoform of yeast glyceraldehyde-3-phosphate dehydrogenase is degraded via this pathway. This import and degradation pathway is reminiscent of the protein transport pathway from the cytosol to lysosomes of mammalian cells.

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“…The propeptide contains information sufficient for vacuolar sorting which occur in a late Golgi compartment. However, upon over-expression, PrA is also secreted into the growth medium, where it accumulates in the mature form Wolff et al, 1996). We found that YAL048c expressed from a multicopy plasmid caused enhanced secretion of PrA.…”

Section: Introductionmentioning

confidence: 77%

“…The yeast strains used were isogenic derivatives of JC482 (Cannon and Tatchell, 1987): YNG318 (JC482 pep4-1 leu2-2) (Wolff et al, 1996), JC482a (JC482 MATa), WY29 (YNG318 X YNG318a), and WY30 (JC482 X JC482a). Yeast strains were grown in either standard YPD medium (Sherman, 1991) or in synthetic complete medium (SC), which is the minimal medium described by Zimmermann (1973) supplemented with nutrients at the concentrations given by Sherman (1991).…”

Section: Strains and Mediamentioning

confidence: 99%