In vitro studies on the translocation of acid phosphatase into the endoplasmic reticulum of the yeast Saccharomyces cerevisiae.
We demonstrate here the in vitro translocation of yeast acid phosphatase into rough endoplasmic reticulum. The precursor of the repressible acid phosphatase from Saccharomyces cerevisiae encoded by the PHO5 gene, was synthesized in a yeast lysate programmed with in vitro transcribed PHO5 mRNA. In the presence of yeast rough microsomes up to 16% of the acid phosphatase synthesized was found to be translocated into the microsomes, as judged by proteinase resistance, and fully core-glycosylated. The translocation efficiency however, decreased to 3% if yeast rough microsomes were added after synthesis of acid phosphatase had been terminated. When a wheat-germ extract was used for in vitro synthesis, the precursor of acid phosphatase was translocated into canine pancreatic rough microsomes and thereby core-glycosylated in a signal-recognition-particle-dependent manner. Replacing canine with yeast rough microsomes in the wheat-germ translation system, however, resulted in a significant decrease in the ability to translocate and glycosylate the precursor. Translocation and glycosylation were partially restored by a high-salt extract prepared from yeast ribosomes. The results presented here suggest that yeast-specific factors are needed to translocate and glycosylate acid phosphatase efficiently in vitro.
Krebs
H O
HO
Biochemisches Institut der Universität, Freiburg, Federal Republic of Germany.
Hoffschulte
H K
HK
Müller
M
M
D000135:Acid Phosphatase / Q000235:genetics* / Q000302:isolation & purification; D004721:Endoplasmic Reticulum / Q000201:enzymology*; D005796:Genes; D005800:Genes, Fungal; D006031:Glycosylation; D008861:Microsomes / Q000201:enzymology; D010957:Plasmids; D014176:Protein Biosynthesis; D011499:Protein Processing, Post-Translational; D012270:Ribosomes / Q000201:enzymology; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D014158:Transcription, Genetic
D000135:Acid Phosphatase