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, alpha-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.
Schu
Peter
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Georg-August-University Göttingen, Zentrum für Biochemie und Molekulare Zellbiologie, Biochemie II, Göttingen, Germany.
D000626:Aminopeptidases / Q000378:metabolism*; D001343:Autophagy / Q000502:physiology; D001681:Biological Assay / Q000379:methods*; D001692:Biological Transport / Q000502:physiology; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D014617:Vacuoles / Q000378:metabolism*; D043323:alpha-Mannosidase / Q000378:metabolism
D029701:Saccharomyces cerevisiae Proteins; D043323:alpha-Mannosidase; D000626:Aminopeptidases; C019689:APE1 protein, S cerevisiae