RESUMO
Hsp100 chaperones cooperate with the Hsp70 chaperone system to disaggregate and reactivate heat-denatured aggregated proteins to promote cell survival after heat stress. The homology models of Hsp100 disaggregases suggest the presence of a conserved network of ionic interactions between the first nucleotide binding domain (NBD1) and the coiled-coil middle subdomain, the signature domain of disaggregating chaperones. Mutations intended to disrupt the putative ionic interactions in yeast Hsp104 and bacterial ClpB disaggregases resulted in remarkable changes of their biochemical properties. These included an increase in ATPase activity, a significant increase in the rate of in vitro substrate renaturation, and partial independence from the Hsp70 chaperone in disaggregation. Paradoxically, the increased activities resulted in serious growth impediments in yeast and bacterial cells instead of improvement of their thermotolerance. Our results suggest that this toxic activity is due to the ability of the mutated disaggregases to unfold independently from Hsp70, native folded proteins. Complementary changes that restore particular salt bridges within the suggested network suppressed the toxic effects. We propose a novel structural aspect of Hsp100 chaperones crucial for specificity and efficiency of the disaggregation reaction.
Assuntos
Proteínas de Choque Térmico HSP70/química , Proteínas de Choque Térmico/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Adenosina Trifosfatases/química , Sequência de Aminoácidos , Endopeptidase Clp , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Proteínas de Fluorescência Verde/química , Proteínas de Choque Térmico/metabolismo , Íons , Modelos Moleculares , Conformação Molecular , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Ligação Proteica , Desnaturação Proteica , Estrutura Terciária de Proteína , Proteínas de Saccharomyces cerevisiae/química , Homologia de Sequência de Aminoácidos , Thermus thermophilus/metabolismoRESUMO
Maf1 is a negative regulator of RNA polymerase III (Pol III) in yeast. Maf1-depleted cells manifest elevated tRNA transcription and inability to grow on non-fermentable carbon source, such as glycerol. Using genomic microarray approach, we examined the effect of Maf1 deletion on expression of Pol II-transcribed genes in yeast grown in medium containing glycerol. We found that transcription of FBP1 and PCK1, two major genes controlling gluconeogenesis, was decreased in maf1Δ cells. FBP1 is located on chromosome XII in close proximity to a tRNA-Lys gene. Accordingly we hypothesized that decreased FBP1 mRNA level could be due to the effect of Maf1 on tgm silencing (tRNA gene mediated silencing). Two approaches were used to verify this hypothesis. First, we inactivated tRNA-Lys gene on chromosome XII by inserting a deletion cassette in a control wild type strain and in maf1Δ mutant. Second, we introduced a point mutation in the promoter of the tRNA-Lys gene cloned with the adjacent FBP1 in a plasmid and expressed in fbp1Δ or fbp1Δ maf1Δ cells. The levels of FBP1 mRNA were determined by RT-qPCR in each strain. Although the inactivation of the chromosomal tRNA-Lys gene increased expression of the neighboring FBP1, the mutation preventing transcription of the plasmid-born tRNA-Lys gene had no significant effect on FBP1 transcription. Taken together, those results do not support the concept of tgm silencing of FBP1. Other possible mechanisms are discussed.
Assuntos
Frutose-Bifosfatase/metabolismo , Genes Fúngicos , Gluconeogênese/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismo , Frutose-Bifosfatase/genética , Deleção de Genes , Regulação Fúngica da Expressão Gênica , Mutação , Fosfoenolpiruvato Carboxiquinase (ATP)/genética , Fosfoenolpiruvato Carboxiquinase (ATP)/metabolismo , RNA Polimerase III/metabolismo , RNA Fúngico/genética , RNA Fúngico/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA de Transferência/genética , RNA de Transferência/metabolismo , RNA de Transferência de Lisina/genética , RNA de Transferência de Lisina/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Fatores de Transcrição/genética , Transcrição GênicaRESUMO
Besides its beneficial role in thermotolerance, the chaperone protein Hsp104 is involved in the inheritance of yeast Saccharomyces cerevisiae prions. Guanidine hydrochloride was previously shown to interfere with Hsp104 chaperone activity in vivo, thus impairing thermotolerance and resulting in prion curing. It was also reported that guanidine inhibits Hsp104 ATPase and disaggregation activity. We show that in vitro guanidine significantly inhibits the disaggregation activity of ClpB, the bacterial orthologue of Hsp104. However, guanidine exerts opposite effects on the ATPase activities of Hsp104 and ClpB. While the ATPase activity of Hsp104 is inhibited, the analogous ClpB activity is stimulated several-fold. Mutation of the universally conserved aspartic acid residue in position 184 to serine (D184S) in HSP104 and the analogous mutation in clpB (D178S) resulted in chaperones with lower disaggregating and ATPase activities. The activities of such changed chaperones are not influenced by guanidine, which suggests the role of this residue in the interaction with guanidine.