In vivo and in vitro analysis of ptl1, a yeast ts mutant with a membrane-associated defect in protein translocation.
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.
Toyn
J
J
Department of Biological Chemistry, UCLA School of Medicine 90024.
Hibbs
A R
AR
Sanz
P
P
Crowe
J
J
Meyer
D I
DI
D001693:Biological Transport, Active; D004721:Endoplasmic Reticulum / Q000378:metabolism; D005656:Fungal Proteins / Q000378:metabolism; D005800:Genes, Fungal; D007425:Intracellular Membranes / Q000378:metabolism; D009154:Mutation; D011498:Protein Precursors / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D013696:Temperature
D005656:Fungal Proteins; D011498:Protein Precursors