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In vivo and in vitro analysis of ptl1, a yeast ts mutant with a membrane‐associated defect in protein translocation.
Author(s) -
Toyn J.,
Hibbs A. R.,
Sanz P.,
Crowe J.,
Meyer D. I.
Publication year - 1988
Publication title -
the embo journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 7.484
H-Index - 392
eISSN - 1460-2075
pISSN - 0261-4189
DOI - 10.1002/j.1460-2075.1988.tb03333.x
Subject(s) - endoplasmic reticulum , biology , cytosol , mutant , chromosomal translocation , biochemistry , saccharomyces cerevisiae , microbiology and biotechnology , fusion protein , membrane protein , membrane , yeast , enzyme , recombinant dna , gene
Mutants defective in the ability to translocate proteins across the membrane of the endoplasmic reticulum were selected in Trp‐ Saccharomyces cerevisiae on the basis of their ability to retain a fusion protein in the cytosol. The fusion comprised the prepro region of prepro‐alpha‐factor (MF alpha 1) N‐terminal to phosphoribosyl anthranilate isomerase (TRP1). The first of the protein translocation mutations, called ptl1, results in temperature‐sensitivity of growth and protein translocation. At the non‐permissive temperature, precursors to several secretory proteins accumulate in the cytosol. Using this mutant, we demonstrate that the prepro‐carboxypeptidase Y that had been accumulated in the cytosol at the non‐permissive temperature could be post‐translationally translocated into the endoplasmic reticulum when cells were returned to the permissive temperature. This result indicates that post‐translational translocation of preproteins across endoplasmic reticulum membranes can occur in vivo. We have also determined that the temperature‐sensitive component is membrane‐associated in ptl1, and that the membranes derived from this strain show a reversible temperature‐sensitive translocation phenotype in vitro.