GSA11 encodes a unique 208-kDa protein required for pexophagy and autophagy in Pichia pastoris

Cells are capable of adapting to changes in their environment by synthesizi ng needed proteins and degrading superfluous ones. Pichia pastoris synthesi zes peroxisomal enzymes to grow in methanol medium. Upon adapting from meth anol medium to one containing glucose, this yeast rapidly and selectively d egrades peroxisomes by an autophagic process referred to as pexophagy. In t his study, we have utilized a novel approach to identify genes required for this degradative pathway. Our approach involves the random integration of a vector containing the Zeocin resistance gene into the yeast genome by res triction enzyme-mediated integration. Cells unable to degrade peroxisomes d uring glucose adaptation were isolated, and the genes that were disrupted b y the insertion of the vector were determined by sequencing. By using this approach, we have identified a number of genes required for glucose-induced selective autophagy of peroxisomes (GSA genes). We report here the charact erization of Gsa11, a unique 208-kDa protein. We found that this protein is required for glucose-induced pexophagy and starvation-induced autophagy. G sa11 is a cytosolic protein that becomes associated with one or more struct ures situated near the vacuole during glucose adaptation. The punctate loca lization of Gsa11 was not observed in gsa10, gsa12, gsa14, and gsa19 mutant s. We have previously shown that Gsa9 appears to relocate from a compartmen t at the vacuole surface to regions between the vacuole and the peroxisomes being sequestered. In the gsa11 mutants, the vacuole only partially surrou nded the peroxisomes, but Gsa9 was still distributed around the peroxisome cluster. This suggests that Gsa9 binds to the peroxisomes independent of th e vacuole. The data also indicate that Gsa11 is not necessary for Gsa9 to i nteract with peroxisomes but acts at an intermediate event required for the vacuole to engulf the peroxisomes.