APG14P AND APG6 VPS30P FORM A PROTEIN COMPLEX ESSENTIAL FOR AUTOPHAGYIN THE YEAST, SACCHAROMYCES-CEREVISIAE/

Mutation in the Saccharomyces cerevisiae APG14 gene causes a defect in autophagy, Cloning and structural analysis of the APG14 gene revealed that APG14 encodes a novel hydrophilic protein with a predicted molec ular mass of 40.5 kDa, and that Apg14p has a coiled-coil motif at its N terminus region, We found that overproduction of Apg14p partially re versed the defect in autophagy induced by the apg6-1 mutation. The apg 6-1 mutant was found to be defective not only in autophagy but also in sorting of carboxypeptidase Y (CPY), a vacuolar-soluble hydrolase, to the vacuole, However, overexpression of APG14 did not alter the CPY s orting defect of the apg6-1 mutant, nor did the apg14 null mutation af fect the CPY sorting pathway. Structural analysis of APG6 revealed tha t APG6 is identical to VPS30, which is involved in a retrieval step of the CPY receptor, Vps10p, to the late-Golgi from the endosome (Seaman , M. N. J., Marcusson, E. G., Cereghino, J. L., and Emr, S. D. (1997) J. Cell Biol. 137, 79-92), Subcellular fractionation indicated that Ap g14p and Apg6p peripherally associated with a membrane structure(s), A pg14p was co-immunoprecipitated with Apg6p, suggesting that they form a stable protein complex. These results imply that Apg6/Vps30p has two distinct functions in the autophagic process and the vacuolar protein sorting pathway. Apg14p may be a component specifically required for the function of Apg6/Vps30p through the autophagic pathway.