GLUCOSE-INDUCED MICROAUTOPHAGY IN PICHIA-PASTORIS REQUIRES THE ALPHA-SUBUNIT OF PHOSPHOFRUCTOKINASE

We have characterized biochemically, morphologically, and genetically two distinct pathways for the selective degradation of peroxisomes in Pichia pastoris. These pathways are independently regulated and analog ous to microautophagy and macroautophagy that have been defined in mam malian cells, When P. pastoris is grown in methanol, cytosolic and per oxisomal enzymes necessary for methanol assimilation are synthesized, During adaptation from methanol to glucose, these enzymes are rapidly and selectively degraded within the yeast vacuole by microautophagy, W e have isolated gsa mutants that are defective in glucose-induced sele ctive autophagy of peroxisomes, In this study, we have shown that gsal is unable to sequester peroxisomes into the yeast vacuole, In additio n, we provide evidence that the glucose-induced selective autophagy 1 (GSA1) protein is the alpha subunit of the phosphofructokinase enzyme complex encoded by PFK1. First, we can rescue the gsal mutant by trans formation with a vector containing PFK1, Second, cellular levels of bo th PFK1 mRNA and phosphofructokinase activity are dramatically reduced in gsal when compared to the parental GS115, Third, a PFK1 knockout ( Delta pfk1) is unable to degrade alcohol oxidase during glucose adapta tion, As observed in gsal, the peroxisomes in Delta pfk1 remain outsid e the vacuole during adaptation, Our data are consistent with the conc ept that PFK1 protein is required for an event upstream of vacuole deg radation (i.e. signaling, selection, or sequestration), However, the d egradation of peroxisomes does not require a catalytically active phos phofructokinase. The inability of Delta pfk1 cells to degrade alcohol oxidase can be rescued by transformation with either normal PFK1 or mu tant pfk1 whose catalytic site had been inactivated by a single amino acid mutation, We propose that PFK1 protein directly modulates glucose -induced microautophagy independent of its ability to metabolize gluco se intermediates.