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Hsp70 chaperones assist in protein folding, disaggregation, and membrane translocation by binding to substrate proteins with an ATP-regulated affinity that relies on allosteric coupling between ATP-binding and substrate-binding domains. We have studied single- and two-domain versions of the E. coli Hsp70, DnaK, to explore the mechanism of interdomain(More)
In Escherichia coli, DnaK is essential for the replication of bacteriophage lambda DNA; this in vivo activity provides the basis of a screen for mutations affecting DnaK function. Mn PCR was used to introduce mutations into residues 405-468 of the C-terminal polypeptide-binding domain of DnaK. These mutant proteins were screened for the ability to propagate(More)
ER-associated degradation (ERAD) removes defective and mis-folded proteins from the eukaryotic secretory pathway, but mutations in the ER lumenal Hsp70, BiP/Kar2p, compromise ERAD efficiency in yeast. Because attenuation of ERAD activates the UPR, we screened for kar2 mutants in which the unfolded protein response (UPR) was induced in order to better define(More)
How substrate affinity is modulated by nucleotide binding remains a fundamental, unanswered question in the study of 70 kDa heat shock protein (Hsp70) molecular chaperones. We find here that the Escherichia coli Hsp70, DnaK, lacking the entire alpha-helical domain, DnaK(1-507), retains the ability to support lambda phage replication in vivo and to pass(More)
The ADP/ATP translocator, a transmembrane protein of the mitochondrial inner membrane, is coded in Saccharomyces cerevisiae by the nuclear gene PET9. DNA sequence analysis of the PET9 gene showed that it encoded a protein of 309 amino acids which exhibited a high degree of homology with mitochondrial translocator proteins from other sources. This(More)
The two apocytochrome c proteins of yeast are coded for by separate genes. Iso-2-cytochrome c differs from the iso-1 protein at 17 positions within a homologous sequence of 108 amino acids. The previously cloned iso-1-cytochrome c coding sequence has been used to identify lambda-yeast recombinant phage containing the gene for iso-2-cytochrome c. The latter(More)
DNA from the cloned yeast iso-1-cytochrome c, cycl, gene was used in a hybridization assay to measure levels and rates of synthesis of cycl RNA. Derepressed cells synthesized cycl RNA at 6 times the rate of that of glucose-repressed cells. Upon glucose addition to a derepressed culture, the transcription of the cycl gene was repressed within 2.5 min. The(More)
The iso-1-cytochrome c gene of yeast has been identified and cloned using a synthetic oligodeoxynucleotide as a hybridization probe. The oligomer d[pT-T-A-G-C-A-G-A-A--C-C-G-G] is complementary to a region near the N terminal coding region of the yeast cyc 1 gene. Of several yeast Eco RI fragments which hybridize to this probe, one is changed in size by a G(More)