Human Dolichol Kinase, a Polytopic Endoplasmic Reticulum Membrane Protein with a Cytoplasmically Oriented CTP-binding Site

We have investigated the role of membrane proteins and lipids during early phases of the cotranslational insertion of secretory proteins into the translocation channel of the endoplasmic reticulum (ER) membrane. We demonstrate that all steps, including the one during which signal sequence recognition occurs, can be reproduced with purified translocation components in detergent solution, in the absence of bulk lipids or a bilayer. Photocross-linking experiments with native membranes show that upon complete insertion into the channel signal sequences are both precisely positioned with respect to the protein components of the channel and contact lipids. Together, these results indicate that signal sequences are bound to a specific binding site at the interface between the channel and the surrounding lipids, and are recognized ultimately by protein–protein interactions. Our data also suggest that at least some signal sequences reach the binding site by transfer through the interior of the channel.

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Protein translocation mutants defective in the insertion of integral membrane proteins into the endoplasmic reticulum.

Molecular Biology of the Cell ◽

10.1091/mbc.3.2.129 ◽

1992 ◽

Vol 3 (2) ◽

pp. 129-142 ◽

Cited By ~ 223

Author(s):

C J Stirling ◽

J Rothblatt ◽

M Hosobuchi ◽

R Deshaies ◽

R Schekman

Keyword(s):

Endoplasmic Reticulum ◽

Membrane Protein ◽

Protein Translocation ◽

Secretory Protein ◽

Polyclonal Antiserum ◽

Secretory Proteins ◽

Temperature Sensitive ◽

Endoplasmic Reticulum Membrane ◽

Membrane Spanning ◽

Coa Reductase

Yeast mutants defective in the translocation of soluble secretory proteins into the lumen of the endoplasmic reticulum (sec61, sec62, sec63) are not impaired in the assembly and glycosylation of the type II membrane protein dipeptidylaminopeptidase B (DPAPB) or of a chimeric membrane protein consisting of the multiple membrane-spanning domain of yeast hydroxymethylglutaryl CoA reductase (HMG1) fused to yeast histidinol dehydrogenase (HIS4C). This chimera is assembled in wild-type or mutant cells such that the His4c protein is oriented to the ER lumen and thus is not available for conversion of cytosolic histidinol to histidine. Cells harboring the chimera have been used to select new translocation defective sec mutants. Temperature-sensitive lethal mutations defining two complementation groups have been isolated: a new allele of sec61 and a single isolate of a new gene sec65. The new isolates are defective in the assembly of DPAPB, as well as the secretory protein alpha-factor precursor. Thus, the chimeric membrane protein allows the selection of more restrictive sec mutations rather than defining genes that are required only for membrane protein assembly. The SEC61 gene was cloned, sequenced, and used to raise polyclonal antiserum that detected the Sec61 protein. The gene encodes a 53-kDa protein with five to eight potential membrane-spanning domains, and Sec61p antiserum detects an integral protein localized to the endoplasmic reticulum membrane. Sec61p appears to play a crucial role in the insertion of secretory and membrane polypeptides into the endoplasmic reticulum.

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