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 family of membrane water channels. The NH2- and COOH-terminal halves of CHIP are sequence-related, and hydropathy analysis predicted six membrane-spanning domains with five connecting loops (A-E). Here, we determined 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 inner leaflet of the membrane by alpha-chymotrypsin digestion of intact oocytes or inside-out membrane vesicles. The E1 epitope at Thr-120 was protease-sensitive in intact oocytes, indicating that loop C is exofacial. The E1 epitope at Lys-6, Arg-162, or Lys-267 was protease-sensitive in inside-out membrane vesicles, confirming the cytoplasmic location 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 were consistent with inner (loop B) and outer (loop E) leaflet locations. 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.
Preston
G M
GM
Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
Jung
J S
JS
Guggino
W B
WB
Agre
P
P
D000595:Amino Acid Sequence; D000818:Animals; D051398:Aquaporin 1; D020346:Aquaporins; D001789:Blood Group Antigens; D002462:Cell Membrane / Q000378:metabolism; D002918:Chymotrypsin; D000939:Epitopes / Q000737:chemistry; D004910:Erythrocyte Membrane / Q000378:metabolism; D005260:Female; D006031:Glycosylation; D006801:Humans; D008565:Membrane Proteins / Q000096:biosynthesis / Q000737:chemistry*; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D016297:Mutagenesis, Site-Directed; D009865:Oocytes / Q000502:physiology; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D014982:Xenopus laevis
C496020:AQP1 protein, human; D020346:Aquaporins; D001789:Blood Group Antigens; D000939:Epitopes; D008565:Membrane Proteins; D011994:Recombinant Proteins; D051398:Aquaporin 1; D002918:Chymotrypsin