title: The C-terminal domain, but not the interchain disulphide, is required for the activity and intracellular trafficking of aminopeptidase A.
abstract: Mammalian aminopeptidase A (APA; glutamyl aminopeptidase; EC 3.4.11.7) is a type II membrane-spanning protein consisting of a short N-terminal cytosolic domain, a single transmembrane domain and a large extracellular C-terminal domain containing the active site. The extracellular domain consists of a 107 kDa domain, containing the zinc-binding motif and all the residues involved in catalysis, separated by a protease-susceptible hinge region from the 45 kDA C-terminal domain of unknown function. To investigate the role of the 45 kDa domain, a construct of murine APA (G594Delta) lacking this C-terminal domain was expressed in COS-1 cells. This truncated form of APA, although expressed, lacked enzymic activity and failed to reach the cell surface. Confocal immunofluorescence microscopy revealed that G594Delta co-localized with the lectin concanavalin A and had a similar staining pattern as protein disulphide-isomerase, indicating that it was retained in the endoplasmic reticulum. Thus the C-terminal 45 kDa domain appears to be acting like a pro-domain and seems to be required for the correct folding and trafficking of APA. In contrast, mutation of cysteine-43 to serine, which is involved in the disulphide-linkage of the APA homodimer, did not affect the enzymic activity, cellular location or rate of trafficking through the secretory pathway of APA.
authors.affiliation: Proteolysis Research Group, School of Biochemistry and Molecular Biology, University of Leeds, Leeds LS2 9JT, U.K.
authors.forename: Lisa D
authors.initials: LD
authors.lastname: Ofner
authors.forename: Nigel M
authors.initials: NM
authors.lastname: Hooper
mesh_terms: D000626:Aminopeptidases / Q000378:metabolism*; D000818:Animals; D019556:COS Cells; D004220:Disulfides / Q000737:chemistry / Q000378:metabolism*; D043384:Glutamyl Aminopeptidase; D008856:Microscopy, Fluorescence; D021381:Protein Transport
chemical_list: D004220:Disulfides; D000626:Aminopeptidases; D043384:Glutamyl Aminopeptidase