DIVERGENT MODES OF AUTOPHAGY IN THE METHYLOTROPHIC YEAST PICHIA-PASTORIS

The budding yeast Pichia pastoris responds to methanolic media by synt hesizing high levels of cytosolic enzymes (e.g. formate dehydrogenase) and peroxisomal enzymes (e.g. alcohol oxidase), which are necessary t o assimilate this carbon source, Major alterations in cellular metabol ism are initiated upon a shift in carbon source to ethanol or glucose, These alterations require the synthesis of new proteins and the rapid degradation of those enzymes no longer needed for methanol utilizatio n, In this study, we have measured cytosolic and peroxisomal enzyme ac tivities and examined the fate of morphologically distinct peroxisomes to assess the degradative response of this yeast during nutrient adap tation, Utilizing biochemical, morphological and genetic approaches, w e have shown that there exist in P. pastoris at least two pathways for the sequestration of peroxisomes into the vacuole for degradation, Th e ethanol-induced pathway is independent of protein synthesis and incl udes an intermediate stage in which individual peroxisomes are sequest ered into autophagosomes by wrapping membranes, which then fuse with t he vacuole, This process is analogous to macroautophagy, The glucose-i nduced pathway invokes the engulfment of dusters of peroxisomes by fin ger-like protrusions of the vacuole by a process analogous to microaut ophagy, Unlike ethanol adaptation, glucose stimulated the degradation of formate dehydrogenase as well, Peroxisomes remained outside the vac uoles of glucose-adapted cyclo-heximide-treated normal cells, suggesti ng that protein synthesis is required for peroxisome entry into the ye ast vacuole, Two complementary mutants (gsa1 and gsa2) that are unable to degrade peroxisomes or formate dehydrogenase during glucose adapta tion were isolated, The mutated gene products appear to function in on e or more events upstream of degradation within the vacuole, since eth anol-induced peroxisome degradation proceeded normally in these mutant s and peroxisomes were found outside the vacuoles of glucose-adapted g sa2 cells, Mutants lacking vacuolar proteinases A and B were unable to degrade alcohol oxidase or formate dehydrogenase during ethanol or gl ucose adaptation, Peroxisomes were found to accumulate within the vacu oles of these proteinase mutants during adaptation, Combined, the resu lts suggest that there exist in Pichia pastoris two independent pathwa ys for the sequestration of peroxisomes into the vacuole, the site of degradation.