Chapter 6 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, α‐mannosidase, is also transported by direct binding to the Atg19 receptor and to aminopeptidase I. Aminopeptidase 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.