BIOGENESIS AND TRANSMEMBRANE TOPOLOGY OF THE CHIP28 WATER CHANNEL AT THE ENDOPLASMIC-RETICULUM

Citation
Wr. Skach et al., BIOGENESIS AND TRANSMEMBRANE TOPOLOGY OF THE CHIP28 WATER CHANNEL AT THE ENDOPLASMIC-RETICULUM, The Journal of cell biology, 125(4), 1994, pp. 803-815
Citations number
59
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Cytology & Histology
Journal title
ISSN journal
0021-9525
Volume
125
Issue
4
Year of publication
1994
Pages
803 - 815
Database
ISI
SICI code
0021-9525(1994)125:4<803:BATTOT>2.0.ZU;2-E
Abstract
CHIP28 is a 28-kD hydrophobic integral membrane protein that functions as a water channel in erythrocytes and renal tubule epithelial cell m embranes. We examined the transmembrane topology of CHIP28 in the ER b y engineering a reporter of translocation (derived from bovine prolact in) into nine sequential sites in the CHIP28 coding region. The result ing chimeras were expressed in Xenopus oocytes, and the topology of th e reporter with respect to the ER membrane was determined by protease sensitivity. We found that although hydropathy analysis predicted up t o seven potential transmembrane regions, CHIP28 spanned the membrane o nly four times. Two putative transmembrane helices, residues 52-68 and 143-157, reside on the lumenal and cytosolic surfaces of the ER membr ane, respectively. Topology derived from these chimeric proteins was s upported 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 tha t direct events of CHIP28 topogenesis. A signal sequence located withi n 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 intern al signal sequence, residues 155-186, reinitiated translocation of a C OOH-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 region s. From this data, we propose a structural model for CHIP28 at the ER membrane in which four membrane-spanning alpha-helices form a central aqueous channel through the lipid bilayer and create a pathway for wat er transport.