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Overexpression of a rat kinase-deficient phosphoinositide 3-kinase, Vps34p, inhibits cathepsin D maturation
Paula Row, Barbara J. REAVES, Jan DOMIN, J. Paul LUZIO, Howard W. DAVIDSON
Biochemical Journal, Volume: 353, Issue: 3, Pages: 655 - 661
Swansea University Author: Paula Row
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DOI (Published version): 10.1042/bj3530655
Lipid kinases and their phosphorylated products are important regulators of many cellular processes, including intracellular membrane traffic. The best example of this is provided by the class III phosphoinositide 3-kinase (PI-3K), Vps34p, which is required for correct targeting of newly synthesized...
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Lipid kinases and their phosphorylated products are important regulators of many cellular processes, including intracellular membrane traffic. The best example of this is provided by the class III phosphoinositide 3-kinase (PI-3K), Vps34p, which is required for correct targeting of newly synthesized carboxy- peptidase Y to the yeast vacuole. A probable mammalian Vps34p orthologue has been previously identified, but its function in the trafficking of lysosomal enzymes has not been resolved. To investigate the possible role(s) of mammalian Vps34p in protein targeting to lysosomes, we have cloned the rat orthologue and overexpressed a kinase-deficient mutant in HeLa cells. Expression of the mutant protein inhibited both maturation of procathepsin D and basal secretion of the precursor. In contrast wortmannin,INTRODUCTIONThere is now abundant evidence to indicate that, in addition to their roles in cell signalling, lipid kinases and their phosphorylated products are important regulators of intracellular membrane traffic (reviewed in [1–3]). Perhaps the best example of this is provided by the class III phosphoinositide 3-kinase (PI-3K), Vps34p. This enzyme was first identified as a component of the yeast vacuolar protein sorting machinery, which, when inacti- vated, causes aberrant secretion of newly synthesized carboxy- peptidase Y (CPY) from the yeast Golgi [4,5]. In addition to its role in biosynthetic traffic, there is also considerable evidence to support a requirement for Vps34p in the endocytic pathway. For example, yeast expressing end12 (a mutant allele of VPS34) are defective in transferring endocytosed α-factor to the vacuole , and Vac1p\Vps19p, which is required for fusion of transport vesicles to yeast endosomes, binds the lipid product of Vps34p [7,8]. Vac1p is one member of a family of proteins that contain the FYVE domain , which is implicated in PtdIns(3)P binding [9,10]. Other yeast family members include Vps27p and the PtdIns(3)P 5-kinase Fab1p. Deletion of FAB1 causes abnormally large vacuoles containing fewer internal vesicles than normal to form, suggesting that the lipid product is required for correct sorting in the yeast multivesicular body . Vps34p is the sole PI-3K in Saccharomyces cereisiae , which implies that this enzyme has multiple roles throughout the yeast endocytic system.In general, the membrane trafficking machinery appears highly conserved between all eukaryotes. Hence the involvement of a Vps34p orthologue in targeting newly synthesized lysosomal hydrolases has been postulated on the basis that PI-3K inhibitors,which also inhibited maturation, caused hypersecretion of the precursor. We propose that mammalian Vps34p plays a direct role in targeting lysosomal enzyme precursors to the endocytic pathway in an analogous fashion to its role in the fusion of early endocytic vesicles with endosomes. We further suggest that inhibition of a wortmannin-sensitive enzyme, other than mam- malian Vps34p, is responsible for the failure to recycle unoccupied mannose 6-phosphate receptors to the trans-Golgi network, and consequent hypersecretion of lysosomal enzyme precursors observed in the presence of this drug.
endosome,lysosome,phosphoinositide3-kinase, trans-Golgi network, wortmannin.
Faculty of Medicine, Health and Life Sciences