Secretory protein translocation in a yeast cell-free system can occur posttranslationally and requires ATP hydrolysis.
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.
D000255:Adenosine Triphosphate / Q000378:metabolism; D001693:Biological Transport, Active; D002474:Cell-Free System; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D005656:Fungal Proteins / Q000378:metabolism*; D006023:Glycoproteins / Q000096:biosynthesis / Q000378:metabolism; D000072235:Mating Factor; D008564:Membrane Potentials; D010455:Peptides / Q000378:metabolism*; D014176:Protein Biosynthesis; D011499:Protein Processing, Post-Translational; D012270:Ribosomes / Q000378:metabolism; D012441:Saccharomyces cerevisiae
D005656:Fungal Proteins; D006023:Glycoproteins; D010455:Peptides; D000072235:Mating Factor; D000255:Adenosine Triphosphate