Michael G. Douglas

DNAJ-LIKE PROTEINS : MOLECULAR CHAPERONES AND SPECIFIC REGULATORS OF HSP70

The folding of proteins and the assembly of protein complexes within subcompartments of the eukaryotic cell is catalysed by different members of the Hsp70 protein family. The chaperone function of Hsp70 proteins in these events is regulated by members of the DnaJ-like protein family, which occurs through direct interaction of different Hsp70 and DnaJ-like protein pairs that appear to be specifically adapted to each other. This review highlights the diversity of functions of DnaJ-like proteins by using specific examples of DnaJ-Hsp70 interactions with polypeptides in yeast protein-biogenesis pathways.

YDJ1p facilitates polypeptide translocation across different intracellular membranes by a conserved mechanism

The role of S. cerevisiae YDJ1 protein (YDJ1p) in polypeptide translocation across membranes has been examined. A conditional ydj1 mutant strain (ydj1-151TS) is defective for import of several polypeptides into mitochondria and alpha factor into the endoplasmic reticulum at 37 degrees C. These defects are suppressed by E. coli dnaJ or overexpression of S. cerevisiae SIS1 proteins. A different ydj1 mutant, which cannot be farnesylated (ydj1-S406), displays similar transport defects to the ydj1-151 strain. Furthermore, the ability of purified ydj1-151p to stimulate the ATPase activity of hsp70SSA1 was greatly diminished compared with the wild-type protein. Together, these data suggest that YDJ1p functions in polypeptide translocation in a conserved manner, probably acting at organelle membranes and in association with hsp70 proteins.

ATP13, a nuclear gene of Saccharomyces cerevisiae essential for the expression of subunit 9 of the mitochondrial ATPase

The respiratory deficient nuclear mutant of Saccharomyces cerevisiae, N9‐168, assigned to complementation group G95 was previously shown to lack subunit 9, one of the three mitochondrially encoded subunits of the F0 component of the mitochondrial ATPase. As a consequence of the structural defect in F0, the ATPase activity of G95 mutants is not inhibited by rutamycin. The absence of subunit 9 in N9‐168 has been correlated with a lower steady‐state level of its mRNA and an increase in higher molecular weight precursor transcripts. These results suggest that the mutation is most likely to affect either translation of the ??? mRNA or processing or the primary transcript. We have isolated a nuclear gene, designated ATP13, which complements the respiratory defect and restores rutamycin‐sensitive ATPase in G95 mutants. Disruption of ATP13 induces a respiratory deficiency which is not complemented by G95 mutants. The nucleotide sequence of ATP13 indicates a primary translation product with an M??? of 42 897. The protein has a basic amino terminal signal sequence that is cleaved upon import into mitochondria. No significant primary structure homology is detected with any protein in the most recent libraries.

Cytosolic Reactions in Mitochondrial Protein Import

Cytosolic hsp70 molecules are involved in maintenance of pre-proteins in transport competent conformations. Genetic and biochemical studies in procaryotes indicate that hsp70 (DnaK) functionally interacts with two other cytosolic proteins, DnaJ and GrpE (Ang et al., 1991). Recently, several eukaryotic DnaJ homologs have been identified. We have purified cytosolic hsp70 (SSA1p) and DnaJ (YDJ1p) homologs from S. cerevisiae and characterized interactions between them. SSA1p exhibited a weak ATPase activity which was stimulated about 10 fold by YDJ1p. Stable complex formation between SSA1p and a mitochondria) presequence peptide was demonstrated. Significant reductions in complex formation were only observed in the presence of both ATP and YDJ1 p. Thus, an eukaryotic dnaJ homolog functionally interacts an eukaryotic hsp70 family member to regulate affinity of the chaperone for a polypeptide substrate.