ABSTRACT
Phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P2), made by Fab1p, is essential for vesicle recycling from vacuole/lysosomal compartments and for protein sorting into multivesicular bodies. To isolate PtdIns(3,5)P2 effectors, we identified Saccharomyces cerevisiae mutants that display fab1delta-like vacuole enlargement, one of which lacked the SVP1/YFR021w/ATG18 gene. Expressed Svp1p displays PtdIns(3,5)P2 binding of exquisite specificity, GFP-Svp1p localises to the vacuole membrane in a Fab1p-dependent manner, and svp1delta cells fail to recycle a marker protein from the vacuole to the Golgi. Cells lacking Svp1p accumulate abnormally large amounts of PtdIns(3,5)P2. These observations identify Svp1p as a PtdIns(3,5)P2 effector required for PtdIns(3,5)P2-dependent membrane recycling from the vacuole. Other Svp1p-related proteins, including human and Drosophila homologues, bind PtdIns(3,5)P2 similarly. Svp1p and related proteins almost certainly fold as beta-propellers, and the PtdIns(3,5)P2-binding site is on the beta-propeller. It is likely that many of the Svp1p-related proteins that are ubiquitous throughout the eukaryotes are PtdIns(3,5)P2 effectors. Svp1p is not involved in the contributions of FAB1/PtdIns(3,5)P2 to MVB sorting or to vacuole acidification and so additional PtdIns(3,5)P2 effectors must exist.
Subject(s)
Endosomes/metabolism , Phosphatidylinositol Phosphates/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Vacuoles/metabolism , Amino Acid Sequence , Autophagy-Related Proteins , Base Sequence , Cloning, Molecular , Escherichia coli , Gene Components , Genetic Vectors , Green Fluorescent Proteins/metabolism , Membrane Proteins , Molecular Sequence Data , Phosphotransferases (Alcohol Group Acceptor)/metabolism , Plasmids/genetics , Protein Binding , Protein Folding , Protein Transport/physiology , Rhinovirus , Saccharomyces cerevisiae Proteins/genetics , Sequence Alignment , Sequence Analysis, DNAABSTRACT
BACKGROUND: The PtdIns3P 5-kinase Fab1 makes PtdIns(3,5)P(2), a phosphoinositide essential for retrograde trafficking between the vacuole/lysosome and the late endosome and also for trafficking of some proteins into the vacuole via multivesicular bodies (MVB). No regulators of Fab1 were identified until recently. RESULTS: Visual screening of the Eurofan II panel of S. cerevisiae deletion mutants identified YLR386w as a novel regulator of vacuolar function. Others recently identified this ORF as encoding the vacuolar inheritance gene VAC14. Like fab1 mutants, yeast lacking Vac14 have enlarged vacuoles that do not acidify correctly. FAB1 overexpression corrects these defects. vac14Delta cells make very little PtdIns(3,5)P(2), and hyperosmotic shock does not stimulate PtdIns(3,5)P(2) synthesis in the normal manner, implicating Vac14 in Fab1 regulation. We also show that, like fab1Delta mutants, vac14Delta cells fail to sort GFP-Phm5 to the MVB and thence to the vacuole: irreversible ubiquitination of GFP-Phm5 overcomes this defect. In the BY4742 genetic background, loss of Vac14 causes much more penetrant effects on phosphoinositide metabolism and vacuolar trafficking than does loss of Vac7, another regulator of Fab1. Vac14 contains motifs suggestive of a role in protein trafficking and interacts with several proteins involved in clathrin-mediated membrane sorting and phosphoinositide metabolism. CONCLUSIONS: Vac14 and Vac7 are both upstream activators of Fab1-catalysed PtdIns(3,5)P(2) synthesis, with Vac14 the dominant contributor to the hierarchy of control. Vac14 is essential for the regulated synthesis of PtdIns(3,5)P(2), for control of trafficking of some proteins to the vacuole lumen via the MVB, and for maintenance of vacuole size and acidity.
Subject(s)
Membrane Proteins/physiology , Phosphatidylinositol Phosphates/biosynthesis , Phosphotransferases (Alcohol Group Acceptor)/physiology , Saccharomyces cerevisiae Proteins/physiology , Base Sequence , DNA Primers , Green Fluorescent Proteins , Luminescent Proteins/metabolism , Membrane Proteins/metabolism , Protein Transport , Recombinant Fusion Proteins/metabolism , Saccharomyces cerevisiae Proteins/metabolismABSTRACT
The pH-regulated expression of the acid (AXP) and alkaline (AEP) extracellular proteases of the yeast Yarrowia lipolytica 148 was analysed. Expression in batch and continuous cultures was determined at the mRNA level by Northern blotting, and at the enzyme level by enzyme assays and Western blotting. Culture pH regulated AEP and AXP expression predominantly at the level of mRNA content. Highest levels of AEP mRNA were detected at pH 6.5 whereas highest levels of AXP mRNA were detected at pH 5.5. At pH values either side of these maxima AEP and AXP expression were progressively down-regulated. For both enzymes, the variation in mRNA levels with culture pH occurred progressively rather than by discrete steps. AXP expression did not occur above pH 7.0. Some degree of AEP expression occurred at all pH values tested in two unrelated strains of Y. lipolytica.
