HEAT-SHOCK TRANSIENTLY ENHANCES THE SYNTHESIS RATE OF SIS1P, A RIBOSOME-ASSOCIATED DNAJ PROTEIN IN THE OLEAGENOUS YEAST APIOTRICHUM-CURVATUM

DnaJ proteins have been localized in different intracellular compartme nts of eukaryotes. In Apiotrichum curvatum, a fat-storing yeast, we fo und a DnaJ homolog associated with ribosomes and large cytosolic compl exes as well. Using a plant DnaJ probe and a cDNA library constructed from poly(A)(+)-RNA of A. curvatum grown on oleate we isolated a SIS1 cDNA coding for a 39.5 kDa protein. The putative protein contains neit her a zinc finger motif nor a CAAX motif but is characterized by a J-d omain at the N-terminal region and a large G-rich region in the middle part of the molecule. Heat shock applied for 1 h resulted in a pronou nced but transient increase of the SIS1 mRNA. An antiserum was raised against the bacterially expressed protein. Cell fractions from A. curv atum were further separated by sedimentation centrifugation on sucrose gradients. Analysing the sub-fractions, we detected Sis1p mainly asso ciated with ribosomes, and with particles sedimenting at approximately 200S. Hsp70 was found to be associated with the 200S fraction. The re spective cytosolic ii. curvatum Hsp70 cDNA was cloned and sequenced. H igh salt conditions caused the removal of Hsp70 and Sis Ip from the 20 0S complexes. Mild RNase treatment of the 200S fraction afforded monos omes and 200S complexes unaffected by RNase. Heat shock led to a prono unced increase in the rate of de novo synthesis. However, due to the l arge pools of Sis1p on ribosomes and large cytosolic complexes, the in crease in gene activation did not lead to a significant change of the total amount of Sis Ip. Accession numbers are: Y12079 for ACHSP70 and Y12080 for ACSIS1. (C) 1998 John Wiley & Sons, Ltd.