Membrane topology of aquaporin CHIP. Analysis of functional epitope-scanning mutants by vectorial proteolysis

摘要: CHIP is the archetypal member of aquaporins, a widely expressed family membrane water channels. The NH2- and COOH-terminal halves are sequence-related, hydropathy analysis predicted six membrane-spanning domains with five connecting loops (A-E). Here, we determined topology in Xenopus oocytes using biologically active recombinant glycosylated at Asn-42, indicating loop A exofacial. An epitope from coronavirus E1 glycoprotein was inserted into localized to outer or inner leaflet by alpha-chymotrypsin digestion intact inside-out vesicles. Thr-120 protease-sensitive oocytes, that C Lys-6, Arg-162, Lys-267 vesicles, confirming cytoplasmic location NH2 COOH termini D. Insertions B E did not produce channels, but their cleavage patterns were consistent (loop B) E) locations. This study indicates functional molecule unique structure two internal repeats oriented 180 degrees each other within membrane.