Interaction of Kar2p and Sls1p is required for efficient co-translational translocation of secreted proteins in the yeast Yarrowia lipolytica.
The yeast Yarrowia lipolytica is a model organism for in vivo study of the signal recognition particle-dependent targeting pathway. In this report, we defined solubilization conditions and set up a fractionation procedure of Y. lipolytica microsomes to determine the amounts of Sec61p-containing translocation pores linked to ribosomes. In contrast to Saccharomyces cerevisiae, from 70 to 80% of Sec61p associates with ribosomes in this yeast. The chaperone protein Kar2p and the Sls1p product, a resident protein of the endoplasmic reticulum lumen, partially fractionate with this Sec61p population. Moreover, Sls1p can be co-immunoprecipitated with Kar2p, and the two polypeptides are shown to directly interact in the yeast two-hybrid system. A site-directed mutagenesis was performed on the SLS1 coding sequence that allowed us to define a functional domain in Sls1p. Indeed, co-translational translocation of a reporter protein is affected when one of these mutant proteins is expressed. Moreover, this protein has lost its capacity to interact with Kar2p, and the two lumenal polypeptides might thus cooperate to promote secretory protein co-translational translocation.
Boisramé
A
A
Laboratoire de Génétique Moléculaire et Cellulaire, INRA, CNRS, Institut National Agronomique Paris-Grignon, 78850 Thiverval-Grignon, France. boisrame@cardere.grignon.inra.fr
Kabani
M
M
Beckerich
J M
JM
Hartmann
E
E
Gaillardin
C
C
D000595:Amino Acid Sequence; D001692:Biological Transport; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000378:metabolism; D005656:Fungal Proteins / Q000378:metabolism*; D005816:Genetic Complementation Test; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism; D008861:Microsomes / Q000378:metabolism; D024101:Mitochondrial Proteins; D008969:Molecular Sequence Data; D009154:Mutation; D010641:Phenotype; D011485:Protein Binding; D014176:Protein Biosynthesis; D012270:Ribosomes / Q000378:metabolism; D000069816:SEC Translocation Channels; D029701:Saccharomyces cerevisiae Proteins; D004718:Saccharomycetales; D017386:Sequence Homology, Amino Acid; D013696:Temperature
D002352:Carrier Proteins; D005656:Fungal Proteins; D018840:HSP70 Heat-Shock Proteins; C060153:KAR2 protein, yeast; D008565:Membrane Proteins; D024101:Mitochondrial Proteins; D000069816:SEC Translocation Channels; C492046:SLS1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins