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

Gregory M. Prestoni, Jin S. Jung, William B Guggino, Peter C Agre

Research output: Contribution to journalArticle

Abstract

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 α-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° to each other within the membrane.

Original languageEnglish (US)
Pages (from-to)1668-1673
Number of pages6
JournalJournal of Biological Chemistry
Volume269
Issue number3
StatePublished - Jan 21 1994

Fingerprint

Aquaporin 1
Proteolysis
Epitopes
Topology
Membranes
Scanning
Aquaporins
Oocytes
Peptide Hydrolases
Cytoplasmic Vesicles
Coronavirus
Chymotrypsin
Xenopus
Ion Channels
Digestion
Glycoproteins
Molecules

ASJC Scopus subject areas

  • Biochemistry

Cite this

Membrane topology of aquaporin CHIP : Analysis of functional epitope-scanning mutants by vectorial proteolysis. / Prestoni, Gregory M.; Jung, Jin S.; Guggino, William B; Agre, Peter C.

In: Journal of Biological Chemistry, Vol. 269, No. 3, 21.01.1994, p. 1668-1673.

Research output: Contribution to journalArticle

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