A mutant of 7SL RNA in Yarrowia lipolytica affecting the synthesis of a secreted protein.
The yeast Yarrowia lipolytica contains two genes, SCR1 and SCR2, encoding a 7SL RNA associated with a signal recognition particle (SRP). To study 7SL RNA function in vivo we have systematically substituted the two conserved nucleotides G128 and A130 in loop 1 of SCR2-encoded 7SL RNA. All single mutations in either nucleotide have no effect. All double mutations are lethal except for one which results in temperature-sensitive growth. We have studied the synthesis and secretion of an alkaline extracellular protease (AEP) in both wild-type and temperature-sensitive mutant strains. Pulse-chase labeling and immunoprecipitation of this protein show that: 1) total protein synthesis is not affected in the mutant strain; 2) levels of AEP precursors in the mutant strain are 60% less than in the wild-type strain; 3) for both strains there is no accumulation of the AEP precursors in the cytoplasm; 4) the kinetics of secretion is similar. These results suggest that mutated SRP is deficient in membrane targeting but still performs translational arrest. This is consistent with the functions of SRP observed in vitro and represents the first demonstration of such roles in vivo.
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Laboratoire de Génétique Institut National de la Recherche Agronomique-Centre, National de la Recherche Scientifique, Thiverval Grignon, France.
Beckerich
J M
JM
Gaillardin
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D001483:Base Sequence; D015152:Blotting, Northern; D005656:Fungal Proteins / Q000096:biosynthesis* / Q000302:isolation & purification; D005800:Genes, Fungal; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D012331:RNA, Fungal / Q000235:genetics* / Q000302:isolation & purification; D012261:Ribonucleoproteins / Q000378:metabolism*; D004718:Saccharomycetales / Q000235:genetics* / Q000378:metabolism; D018271:Signal Recognition Particle; D014170:Transformation, Genetic
D005656:Fungal Proteins; D012331:RNA, Fungal; D012261:Ribonucleoproteins; D018271:Signal Recognition Particle