| Title: | A homologous cell-free system for studying protein translocation across the endoplasmic reticulum membrane in fission yeast |
| Address: | "Department of Biochemistry, University of Illinois at Urbana-Champaign 61801" |
| ISSN/ISBN: | 0749-503X (Print) 0749-503X (Linking) |
| Abstract: | "We report the development of a homologous in vitro assay system for analysing translocation of proteins across the endoplasmic reticulum (ER) membrane of the fission yeast Schizosaccharomyces pombe. Our protocol for preparing an S. pombe extract capable of translating natural messenger RNAs was modified from a procedure previously used for Saccharomyces cerevisiae, in which cells are lysed in a bead-beater. However, we were unable to prepare fission yeast microsomes active in protein translocation using existing budding yeast protocols. Instead, our most efficient preparations were isolated by fractionating spheroplasts, followed by extensive washing and size exclusion chromatography of the crude membranes. Translocation of two ER-targeted proteins, pre-acid phosphatase from S. pombe and prepro-alpha-factor from S. cerevisiae, was monitored using two distinct assays. First, evidence that a fraction of both proteins was sequestered within membrane-enclosed vesicles was provided by resistance to exogenously added protease. Second, the protected fraction of each protein was converted to a higher molecular weight, glycosylated form; attachment of carbohydrate to the translocated proteins was confirmed by their ability to bind Concanavalin A-Sepharose. Finally, we examined whether proteins could be translocated across fission yeast microsomal membranes after their synthesis was complete. Our results indicate that S. cerevisiae prepro-alpha-factor can be post-translationally imported into the fission yeast ER, while S. pombe pre-acid phosphatase crosses the membrane only by a co-translational mechanism" |
| Keywords: | Acid Phosphatase/*metabolism Biological Transport/drug effects Cell Compartmentation Cell Fractionation Cell-Free System Endoplasmic Reticulum/*metabolism Glycosylation Mating Factor Microsomes/metabolism Peptides/*metabolism Protein Biosynthesis Protein; |
| Notes: | "MedlineBrennwald, P Wise, J A eng R01 GM34891/GM/NIGMS NIH HHS/ Comparative Study Research Support, U.S. Gov't, Non-P.H.S. Research Support, U.S. Gov't, P.H.S. England 1994/02/01 Yeast. 1994 Feb; 10(2):159-72. doi: 10.1002/yea.320100204" |
