AMINOPEPTIDASE I ENZYMATIC ACTIVITY

Aminopeptidase I is the cargo protein of the cytoplasm-to-vacuole targeting (Cvt), autophagy-like protein-targeting pathway of the yeast Saccharomyces cerevisiae, the nonclassical vacuolar biosynthetic transport route. The second enzyme following this route to the vacuole, a-mannosidase, is also transported by direct binding to the Atg19 receptor and to aminopeptidase I. Aminopepticlase I forms a homododecameric complex, which is synthesized and assembled in the cytoplasm, packed in double-membrane vesicles, and transported to the vacuole. Only the homododecameric complex of aminopeptidase I has exopeptidase activity directed against amino-terminal leucine residues. Enzymatic activity can be determined spectrofluorometrically in homogenates and semi-quantitatively after nondenaturing gel electrophoresis and by yeast colony-overlay assay. This chapter describes the methods to determine aminopeptidase I enzymatic activity used to follow complex assembly and vacuolar transport.