Isolation and cloning of the Yarrowia lipolytica SEC65 gene, a component of the yeast signal recognition particle displaying homology with the human SRP19 gene.
The signal recognition particle (SRP) is a ribonucleoprotein composed of a 7SL RNA and six polypeptides. Here we report the results of a series of experiments carried out to define the function of the Yarrowia lipolytica homologue of the 19 kDa subunit of mammalian SRP. The YlSEC65 gene product is a 310 amino acid protein. Coimmuneprecipitation of Sec65p and 7SL RNA in Y. lipolytica revealed that these components are stable associated in a complex. Deletion of the YlSEC65 gene is lethal, in contrast with the results described for the Saccharomyces cerevisiae SEC65 gene, which is not essential for cell growth and whose deletion results in slowly growing strains. Using site-directed mutagenesis we demonstrate that the two arginine residues of the EGRR motif conserved in all SRP19 homologues are essential for SRP activity. By random mutagenesis of YlSEC65, we have isolated a temperature-sensitive mutant and shown that it was affected in protein secretion at the non-permissive temperature. We also show that the YlSEC65 gene is able to functionally complement the temperature-sensitive growth of S. cerevisiae sec65 mutants. Our results suggest that SRP-dependent targeting may be the main secretory pathway in Y lipolytica, as has been described for higher eukaryotes.
Sánchez
M
M
Departamento de Microbiología y Genética, Edificio Departamental, Universidad de Salamanca, Spain.
Beckerich
J M
JM
Gaillardin
C
C
Domínguez
A
A
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000276:immunology; D001483:Base Sequence; D003001:Cloning, Molecular; D004271:DNA, Fungal; D010450:Endopeptidases / Q000378:metabolism; D005656:Fungal Proteins / Q000235:genetics* / Q000378:metabolism; D005800:Genes, Fungal; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011817:Rabbits; D012441:Saccharomyces cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D004718:Saccharomycetales / Q000235:genetics* / Q000254:growth & development; D017386:Sequence Homology, Amino Acid; D018271:Signal Recognition Particle / Q000235:genetics*
D000906:Antibodies; D004271:DNA, Fungal; D005656:Fungal Proteins; C489551:SEC65 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D018271:Signal Recognition Particle; D010450:Endopeptidases