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Jill L Johnson

Publications and source records attributed to Jill L Johnson.

4 recordsLinked to original sources
PubMed2006

Nucleotide-dependent interaction of Saccharomyces cerevisiae Hsp90 with the cochaperone proteins Sti1, Cpr6, and Sba1.

The ATP-dependent molecular chaperone Hsp90 and partner cochaperone proteins are required for the folding and activity of diverse cellular client proteins, including steroid hormone receptors and multiple oncogenic kinases. Hsp90 undergoes nucleotide-dependent conformational changes, but little is known about how these changes are coupled to client protein activation. In order to clarify how nucleotides affect Hsp90 interactions with cochaperone proteins, we monitored assembly of wild-type and mutant Hsp90 with Sti1, Sba1, and Cpr6 in Saccharomyces cerevisiae cell extracts. Wild-type Hsp90 bound Sti1 in a nucleotide-independent manner, while Sba1 and Cpr6 specifically and independently interacted with Hsp90 in the presence of the nonhydrolyzable analog of ATP, AMP-PNP. Alterations in Hsp90 residues that contribute to ATP binding or hydrolysis prevented or altered Sba1 and Cpr6 interaction; additional alterations affected the specificity of Cpr6 interaction. Some mutant forms of Hsp90 also displayed reduced Sti1 interaction in the presence of a nucleotide. These studies indicate that cycling of Hsp90 between the nucleotide-free, open conformation and the ATP-bound, closed conformation is influenced by residues both within and outside the N-terminal ATPase domain and that these conformational changes have dramatic effects on interaction with cochaperone proteins.

Adenylyl Imidodiphosphate↗
PubMed2005

Effect of mutation of the tetratricopeptide repeat and asparatate-proline 2 domains of Sti1 on Hsp90 signaling and interaction in Saccharomyces cerevisiae.

Through simultaneous interactions with Hsp70 and Hsp90 via separate tetratricopeptide repeat (TPR) domains, the cochaperone protein Hop/Sti1 has been proposed to play a critical role in the transfer of client proteins from Hsp70 to Hsp90. However, no prior mutational analysis demonstrating a critical in vivo role for the TPR domains of Sti1 has been reported. We used site-directed mutagenesis of the TPR domains combined with a genetic screen to isolate mutations that disrupt Sti1 function. A single amino acid alteration in TPR2A disrupted Hsp90 interaction in vivo but did not significantly affect function. However, deletion of a conserved residue in TPR2A or mutations in the carboxy-terminal DP2 domain completely disrupted Sti1 function. Surprisingly, mutations in TPR1, previously shown to interact with Hsp70, were not sufficient to disrupt in vivo functions unless combined with mutations in TPR2B, suggesting that TPR1 and TPR2B have redundant or overlapping in vivo functions. We further examined the genetic and physical interaction of Sti1 with a mutant form of Hsp90, providing insight into the importance of the TPR2A domain of Sti1 in regulating Hsp90 function.

Amino Acid Substitution↗
PubMed2005

Novel interaction of the Hsp90 chaperone machine with Ssl2, an essential DNA helicase in Saccharomyces cerevisiae.

Hsp90 is an essential molecular chaperone that is critical for the activity of diverse cellular proteins. Hsp90 functions with a number of co-chaperone proteins, including Sti1/Hop. We conducted a genetic screen in Saccharomyces cerevisiae to isolate mutations that exhibit enhanced growth defects in the absence of STI1. We obtained mutations in genes encoding components of the Hsp90 chaperone machine, HSC82, CPR7 and YDJ1, and two essential genes, SSL2 and UTP21, not previously linked to Hsp90. Ssl2, the yeast homologue of XPB, is an ATP-dependent DNA helicase that is a component of the TFIIH multiprotein complex and has dual functions in transcription and DNA repair. In order to determine whether Ssl2 function is dependent on Hsp90, we further examined the interaction between Ssl2 and Hsp90. Multiple mutant alleles of SSL2 exhibited a pronounced growth defect when co-expressed with a mutant allele of Hsp90. In addition, isolation of Ssl2 protein resulted in the co-purification of Hsp90 and Sti1, suggesting that Ssl2 and Hsp90 are in the same protein complexes in vivo. These results suggest a novel role for Hsp90 in the essential cellular functions of transcription and DNA repair.

DNA Helicases↗