Biogenesis and transmembrane topology of the CHIP28 water channel at the endoplasmic reticulum

William Skach, Lan Bo Shi, M. Clara Calayag, Antonio Frigeri, Vishwanath R. Lingappa, A. S. Verkman

Research output: Contribution to journalArticle

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Abstract

CHIP28 is a 28-kD hydrophobic integral membrane protein that functions as a water channel in erythrocytes and renal tubule epithelial cell membranes. We examined the transmembrane topology of CHIP28 in the ER by engineering a reporter of translocation (derived from bovine prolactin) into nine sequential sites in the CHIP28 coding region. The resulting chimeras were expressed in Xenopus oocytes, and the topology of the reporter with respect to the ER membrane was determined by protease sensitivity. We found that although hydropathy analysis predicted up to seven potential transmembrane regions, CHIP28 spanned the membrane only four times. Two putative transmembrane helices, residues 52-68 and 143-157, reside on the lumenal and cytosolic surfaces of the ER membrane, respectively. Topology derived from these chimeric proteins was supported by cell-free translation of five truncated CHIP28 cDNAs, by N-linked glycosylation at an engineered consensus site in native CHIP28 (residue His69), and by epitope tagging of the CHIP28 amino terminus. Defined protein chimeras were used to identify internal sequences that direct events of CHIP28 topogenesis. A signal sequence located within the first 52 residues initiated nascent chain translocation into the ER lumen. A stop transfer sequence located in the hydrophobic region from residues 90-120 terminated ongoing translocation. A second internal signal sequence, residues 155-186, reinitiated translocation of a COOH-terminal domain (residues 186-210) into the ER lumen. Integration of the nascent chain into the ER membrane occurred after synthesis of 107 residues and required the presence of two membrane-spanning regions. From this data, we propose a structural model for CHIP28 at the ER membrane in which four membrane- spanning α-helices form a central aqueous channel through the lipid bilayer and create a pathway for water transport.

Original languageEnglish (US)
Pages (from-to)803-815
Number of pages13
JournalJournal of Cell Biology
Volume125
Issue number4
DOIs
StatePublished - May 1994
Externally publishedYes

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Aquaporins
Endoplasmic Reticulum
Membranes
Protein Sorting Signals
Structural Models
Lipid Bilayers
Xenopus
Glycosylation
Prolactin
Membrane Potentials
Oocytes
Epitopes
Membrane Proteins
Proteins
Peptide Hydrolases
Complementary DNA
Erythrocytes
Epithelial Cells
Cell Membrane
Kidney

ASJC Scopus subject areas

  • Cell Biology

Cite this

Skach, W., Shi, L. B., Calayag, M. C., Frigeri, A., Lingappa, V. R., & Verkman, A. S. (1994). Biogenesis and transmembrane topology of the CHIP28 water channel at the endoplasmic reticulum. Journal of Cell Biology, 125(4), 803-815. https://doi.org/10.1083/jcb.125.4.803

Biogenesis and transmembrane topology of the CHIP28 water channel at the endoplasmic reticulum. / Skach, William; Shi, Lan Bo; Calayag, M. Clara; Frigeri, Antonio; Lingappa, Vishwanath R.; Verkman, A. S.

In: Journal of Cell Biology, Vol. 125, No. 4, 05.1994, p. 803-815.

Research output: Contribution to journalArticle

Skach, W, Shi, LB, Calayag, MC, Frigeri, A, Lingappa, VR & Verkman, AS 1994, 'Biogenesis and transmembrane topology of the CHIP28 water channel at the endoplasmic reticulum', Journal of Cell Biology, vol. 125, no. 4, pp. 803-815. https://doi.org/10.1083/jcb.125.4.803
Skach, William ; Shi, Lan Bo ; Calayag, M. Clara ; Frigeri, Antonio ; Lingappa, Vishwanath R. ; Verkman, A. S. / Biogenesis and transmembrane topology of the CHIP28 water channel at the endoplasmic reticulum. In: Journal of Cell Biology. 1994 ; Vol. 125, No. 4. pp. 803-815.
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