PEROXISOME DEGRADATION BY MICROAUTOPHAGY IN PICHIA-PASTORIS - IDENTIFICATION OF SPECIFIC STEPS AND MORPHOLOGICAL INTERMEDIATES

We used the dye mmoniumpropyl)-4-(p-diethylaminophenylhexatrienyl) pyr idinium dibromide (FM4-64) and a fusion protein, consisting of the gre en fluorescent protein appended to the peroxisomal targeting signal, S er-Lys-Leu (SKL), to label the vacuolar membrane and the peroxisomal m atrix, respectively, in living Pichia pastoris cells and followed by f luorescence microscopy the morphological and kinetic intermediates in the vacuolar degradation of peroxisomes by microautophagy and macroaut ophagy. Structures corresponding to the intermediates were also identi fied by electron microscopy. The kinetics of appearance and disappeara nce of these intermediates is consistent with a precursor-product rela tionship between intermediates, which form the basis of a model for mi croautophagy. Inhibitors affecting different steps of microautophagy d id not impair peroxisome delivery to the vacuole via macroautophagy, a lthough inhibition of vacuolar proteases affected the final vacuolar d egradation of green fluorescent protein (S65T mutant version [GFP])-SK L via both autophagic pathways. P. pastoris mutants defective in perox isome microautophagy (pag mutants) were isolated and characterized for the presence or absence of the intermediates. These mutants, comprisi ng 6 complementation groups, support the model for microautophagy. Our studies indicate that the microautophagic degradation of peroxisomes proceeds via specific intermediates, whose generation and/or processin g is controlled by PAG gene products, and shed light on the poorly und erstood phenomenon of peroxisome homeostasis.