Connexin Membrane Protein Biosynthesis Is Influenced by Polypeptide Positioning within the Translocon and Signal Peptidase Access*
Abstract
We reported previously (Falk, M. M., Kumar, N. M., and Gilula, N. B. (1994) J. Cell Biol. 127, 343–355) that the membrane integration of polytopic connexin polypeptides can be accompanied by an inappropriate cleavage that generates amino-terminal truncated connexins. While this cleavage was not detected in vivo, translation in standard cell-free translation/translocation systems resulted in the complete cleavage of all newly integrated connexins. Partial cleavage occurred in heterologous expression systems that correlated with the expression level. Here we report that the transmembrane topology of connexins generated in microsomal membranes was identical to the topology of functional connexins in plasma membranes. Characterization of the cleavage site and reaction showed that the connexins were processed by signal peptidase immediately downstream of their first transmembrane domain in a reaction similar to the removal of signal peptides from pre-proteins. Increasing the length and hydrophobic character of the signal anchor sequence of connexins completely prevented the aberrant cleavage. This result indicates that their signal anchor sequence was falsely recognized and positioned as a cleavable signal peptide within the endoplasmic reticulum translocon, and that this mispositioning enabled signal peptidase to access the cleavage sites. The results provide direct evidence for the involvement of unknown cellular factors in the membrane integration process of connexins.
Footnotes
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↵* This research was supported by National Institutes of Health Grant GM 37904, a grant from the Lucille P. Markey Charitable Trust (to N. B. G.), and a Deutsche Forschungsgemeinschaft fellowship (to M. M. F.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵‡ To whom all correspondence should be addressed: Dept. of Cell Biology, MB6, The Scripps Research Institute, 10550 North Torrey Pines Rd., La Jolla, CA 92037. Tel.: 1-619-784-2352; Fax: 1-619-784-2345; E-mail: MFalk{at}scripps.edu.
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↵1 The abbreviations used are: ER, endoplasmic reticulum; Cx, connexin; PM, plasma membrane; SA, signal anchor; PAGE, polyacrylamide gel electrophoresis; SP, signal peptide; SPase, signal peptidase; TM, transmembrane; wt, wild type; CAPS, 3-(cyclohexylamino)-1-propanesulfonic acid; CHES, 2-(N-cyclohexylamino)ethanesulfonic acid; NEM,N-ethylmaleimide; PL, prolactin.
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↵2 M. Falk, unpublished data.
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- Received November 3, 1997.
- Revision received January 27, 1998.
- The American Society for Biochemistry and Molecular Biology, Inc.
