The TSR1 gene of Yarrowia lipolytica is involved in the signal recognition particle-dependent translocation pathway of secretory proteins.
We have isolated suppressors (tsr1 to tsr5) of the thermosensitive growth of the scr2.II-13 mutation, which affects the stability of the signal recognition particle. The growth of these mutants is largely affected in the SCR2 context at 34 degrees C. We have studied the synthesis and secretion of an alkaline extracellular protease (AEP) in both wild-type and tsr1-1(SCR2(+)) thermosensitive mutant strains. Pulse-chase labeling and immunoprecipitation of this protein showed that the level of AEP precursors in the tsr1-1(SCR2(+)) strain is 70% less than in the wild-type strain under conditions where the global protein synthesis is practically unaffected. This defect was observed as early as 10 min after the shift to nonpermissive temperature. In neither strain was there any effect on the kinetics of secretion, and no cytoplasmic accumulation was detected. We have cloned the TSR1 gene by complementing the thermosensitive phenotype of a tsr1-1(SCR2(+)) mutant. Analysis of the TSR1 DNA sequence revealed an open reading frame of 1383 base pairs, encoding a serine-rich protein of 461 amino acids with an amino-terminal signal peptide, and a membrane-spanning domain of 20 amino acids that could act as a stop transfer signal to ensure membrane localization of Tsr1p. Two homologues of the TSR1 gene were identified in Saccharomyces cerevisiae (YHC8) and Hansenula polymorpha (YLU2). Disruption of the TSR1 gene revealed that it is an essential single-copy gene. The TSR1 gene encodes a single mRNA of 1.5 kilobase pairs. The study of the synthesis and secretion of AEP in the complemented tsr1-1(SCR2(+),TSR1(+)) strain revealed that the TSR1 gene ensures complete recovery of the synthesis defect and thus could encode an important component of the endoplasmic reticulum membrane involved in the early steps of the signal recognition particle-dependent translocation pathway.
Mamoun
C B
CB
Laboratoire de Génétique Moléculaire et Cellulaire, Institut National de la Recherche Agronomique-CNRS, Centre de Biotechnologie Agro-Industrielle Institut National Agronomique Paris-Grignon, 78850 Thiverval-Grignon, France.
Beckerich
J M
JM
Gaillardin
C
C
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004721:Endoplasmic Reticulum / Q000378:metabolism; D005656:Fungal Proteins / Q000378:metabolism*; D005800:Genes, Fungal; D008565:Membrane Proteins / Q000235:genetics; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D015183:Restriction Mapping; D004718:Saccharomycetales / Q000378:metabolism*; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D012697:Serine Endopeptidases / Q000378:metabolism; D018271:Signal Recognition Particle / Q000378:metabolism; D015003:Yeasts / Q000378:metabolism*
D005656:Fungal Proteins; D008565:Membrane Proteins; D018271:Signal Recognition Particle; C118745:TSR1 protein, Yarrowia lipolytica; D012697:Serine Endopeptidases; C067468:Yarrowia lipolytica alkaline extracellular protease