We have investigated the effects of chemical alkylation of microsomal membranes on nascent chain binding and translocation. Assays were conducted using either full-length or truncated preprolactin transcripts in combination with a reconstituted membrane system consisting of proteolyzed rough microsomes and the cytoplasmic domain of the signal recognition particle receptor. Treatment of rough microsomes with N-ethylmaleimide was observed to inhibit preprolactin processing at a site other than the signal recognition particle or the signal recognition particle receptor. As formation of a translocation competent junction between the ribosome/nascent chain complex and the membrane has recently been demonstrated to require GTP (Connolly, T., and R. Gilmore. J. Cell Biol. 1986. 103:2253-2261), the effects of membrane alkylation on this parameter were assessed. N-ethylmaleimide treatment did not inhibit nascent chain targeting or GTP-dependent signal sequence insertion. Translocation of the targeted and inserted nascent chain was, however, blocked. These data indicate (a) that the process of nascent chain translocation is distinct from targeting and signal sequence insertion, and (b) translocation of the peptide chain across the membrane is mediated by an N-ethylmaleimide-sensitive membrane protein component(s). To further substantiate the observation that nascent chain targeting and signal sequence insertion can be distinguished from translocation, the temperature dependencies of the two phenomena were compared. Signal sequence insertion occurred at low temperatures (4 degrees C) and was maximal between 10 and 15 degrees C. Translocation was only observed at higher temperatures and was maximal between 25 and 30 degrees C.

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