We describe an in vitro system with all components derived from the yeast Saccharomyces cerevisiae that can translocate a yeast secretory protein across microsomal membranes. In vitro transcribed prepro-alpha-factor mRNA served to program a membrane-depleted yeast translation system. Translocation and core glycosylation of prepro-alpha-factor were observed when yeast microsomal membranes were added during or after translation. A membrane potential is not required for translocation. However, ATP is required for translocation and nonhydrolyzable analogues of ATP cannot serve as a substitute. These findings suggest that ATP hydrolysis may supply the energy required for translocation of proteins across the endoplasmic reticulum.
Secretory protein translocation in a yeast cell-free system can occur posttranslationally and requires ATP hydrolysis.
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M G Waters, G Blobel; Secretory protein translocation in a yeast cell-free system can occur posttranslationally and requires ATP hydrolysis.. J Cell Biol 1 May 1986; 102 (5): 1543–1550. doi: https://doi.org/10.1083/jcb.102.5.1543
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