A mutation in the signal recognition particle 7S RNA of the yeast Yarrowia lipolytica preferentially affects synthesis of the alkaline extracellular protease: in vivo evidence for translational arrest.
Replacement of the signal recognition particle (SRP) 7S gene (SCR1) on a replicating plasmid with scr1-1 (G to A at 129 and A to T at 131 in the consensus sequence -GNAR- in the loop of domain III) resulted in temperature sensitivity for growth of cells in which both chromosomal SRP 7S RNA genes were deleted. Pulse-chase immunoprecipitation experiments were done after a shift to non-permissive temperature using the major secreted protein the alkaline extracellular protease (AEP) as a reporter molecule. No untranslocated AEP precursor was detected in a strain with scr1-1 on a plasmid, but the amount of the largest AEP precursor (55 kD) immunoprecipitated as a percentage of total protein synthesized was reduced 68% compared to an isogenic strain with SCR1 on the plasmid. The possibility that an untranslocated precursor was synthesized but not detected because of instability was largely eliminated by detection of a 53-kD untranslocated precursor of a mutated AEP (P17M; methionine replaced proline in the second position of the pro-peptide) which chased to the 55-kD translocated AEP precursor. Thus, SRP has a role in the biosynthesis of AEP. Possibly, the scr1-1 mutation does not affect signal recognition or translational arrest but instead results in maintenance of translational arrest of AEP synthesis. The results also suggest that AEP can be translocated in vivo either co-translationally in which SRP is at least involved in biosynthesis or posttranslationally without SRP involvement.
Yaver
D S
DS
Department of Microbiology, University of California, Davis 95616.
Matoba
S
S
Ogrydziak
D M
DM
D000483:Alleles; D000595:Amino Acid Sequence; D001483:Base Sequence; D005800:Genes, Fungal; D008969:Molecular Sequence Data; D009154:Mutation; D014176:Protein Biosynthesis; D011498:Protein Precursors / Q000378:metabolism; D012331:RNA, Fungal / Q000235:genetics*; D012261:Ribonucleoproteins / Q000235:genetics*; D012697:Serine Endopeptidases / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D018271:Signal Recognition Particle; D015003:Yeasts / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development
D011498:Protein Precursors; D012331:RNA, Fungal; D012261:Ribonucleoproteins; D018271:Signal Recognition Particle; D012697:Serine Endopeptidases; C067468:Yarrowia lipolytica alkaline extracellular protease