MEMBRANE TOPOLOGY OF AQUAPORIN CHIP - ANALYSIS OF FUNCTIONAL EPITOPE-SCANNING MUTANTS BY VECTORIAL PROTEOLYSIS

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