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 compartments of eukaryotes. In Apiotrichum curvatum, a fat-storing yeast, we found a DnaJ homolog associated with ribosomes and large cytosolic complexes 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 neither a zinc finger motif nor a CAAX motif but is characterized by a J-domain 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 pronounced but transient increase of the SIS1 mRNA. An antiserum was raised against the bacterially expressed protein. Cell fractions from A. curvatum were further separated by sedimentation centrifugation on sucrose gradients. Analysing the sub-fractions, we detected Sis1p mainly associated with ribosomes, and with particles sedimenting at approximately 200S. Hsp70 was found to be associated with the 200S fraction. The respective cytosolic A. curvatum Hsp70 cDNA was cloned and sequenced. High salt conditions caused the removal of Hsp70 and Sis1p from the 200S complexes. Mild RNase treatment of the 200S fraction afforded monosomes and 200S complexes unaffected by RNase. Heat shock led to a pronounced increase in the rate of de novo synthesis. However, due to the large pools of Sis1p on ribosomes and large cytosolic complexes, the increase in gene activation did not lead to a significant change of the total amount of Sis1p.
Specht
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V
Fachbereich Chemie, Philipps-Universität, Marburg, Germany.
Lubeck
M
M
Kindl
H
H
D000595:Amino Acid Sequence; D002175:Candida / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D002499:Centrifugation, Density Gradient; D002846:Chromatography, Affinity; D003600:Cytosol / Q000737:chemistry; D018076:DNA, Complementary / Q000302:isolation & purification; D005656:Fungal Proteins / Q000032:analysis / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D050956:HSP40 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins / Q000032:analysis; D006360:Heat-Shock Proteins; D018869:Heat-Shock Response; D008969:Molecular Sequence Data; D014176:Protein Biosynthesis; D012270:Ribosomes / Q000737:chemistry*; D029701:Saccharomyces cerevisiae Proteins; D017422:Sequence Analysis, DNA; D013347:Subcellular Fractions
D018076:DNA, Complementary; D005656:Fungal Proteins; D050956:HSP40 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins; C070320:SIS1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins