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 fractionatio n procedure of Y. lipolytica microsomes to determine the amounts of Se c61p-containing translocation pores linked to ribosomes, In contrast t o Saccharomyces cerevisiae, from 70 to 80% of Sec61p associates with r ibosomes in this yeast. The chaperone protein Kar2p and the Sls1p prod uct, a resident protein of the endoplasmic reticulum lumen, partially fractionate with this Sec61p population, Moreover, Sls1p can be co-imm unoprecipitated with Kar2p, and the two polypeptides are shown to dire ctly interact in the yeast two-hybrid system. A site-directed mutagene sis was performed on the SLS1 coding sequence that allowed us to defin e a functional domain in Sls1p, Indeed, co-translational translocation of a reporter protein is affected when one of these mutant proteins i s expressed. Moreover, this protein has lost its capacity to interact with Kar2p, and the two lumenal polypeptides might thus cooperate to p romote secretory protein co-translational translocation.