Selective Hsp70-Dependent Docking of Hsp104 to Protein Aggregates Protects the Cell from the Toxicity of the Disaggregase.

Hsp104 is a yeast chaperone that rescues misfolded proteins from aggregates associated with proteotoxic stress and aging. Hsp104 consists of N-terminal domain, regulatory M-domain and two ATPase domains, assembled into a spiral-shaped hexamer. Protein disaggregation involves polypeptide extraction from an aggregate and its translocation through the central channel. This process relies on Hsp104 cooperation with the Hsp70 chaperone, which also plays important role in regulation of the disaggregase. Although Hsp104 protein-unfolding activity enables cells to survive stress, when uncontrolled, it becomes toxic to the cell. In this work, we investigated the significance of the interaction between Hsp70 and the M-domain of Hsp104 for functioning of the disaggregation system. We identified phenylalanine at position 508 in Hsp104 to be the key site of interaction with Hsp70. Disruption of this site makes Hsp104 unable to bind protein aggregates and to confer tolerance in yeast cells. The use of this Hsp104 variant demonstrates that Hsp70 allows successful initiation of disaggregation only as long as it is able to interact with the disaggregase. As reported previously, this interaction causes release of the M-domain-driven repression of Hsp104. Now we reveal that, apart from this allosteric effect, the interaction between the chaperone partners itself contributes to effective initiation of disaggregation and plays important role in cell protection against Hsp104-induced toxicity. Interaction with Hsp70 shifts Hsp104 substrate specificity from non-aggregated, disordered substrates toward protein aggregates. Accordingly, Hsp70-mediated sequestering of the Hsp104 unfoldase in aggregates makes it less toxic and more productive.

Molecular cloning and transcriptional activity of a new Petunia calreticulin gene involved in pistil transmitting tract maturation, progamic phase, and double fertilization.

Calreticulin (CRT) is a highly conserved and ubiquitously expressed Ca²âº-binding protein in multicellular eukaryotes. As an endoplasmic reticulum-resident protein, CRT plays a key role in many cellular processes including Ca²âº storage and release, protein synthesis, and molecular chaperoning in both animals and plants. CRT has long been suggested to play a role in plant sexual reproduction. To begin to address this possibility, we cloned and characterized the full-length cDNA of a new CRT gene (PhCRT) from Petunia. The deduced amino acid sequence of PhCRT shares homology with other known plant CRTs, and phylogenetic analysis indicates that the PhCRT cDNA clone belongs to the CRT1/CRT2 subclass. Northern blot analysis and fluorescent in situ hybridization were used to assess PhCRT gene expression in different parts of the pistil before pollination, during subsequent stages of the progamic phase, and at fertilization. The highest level of PhCRT mRNA was detected in the stigma-style part of the unpollinated pistil 1 day before anthesis and during the early stage of the progamic phase, when pollen is germinated and tubes outgrow on the stigma. In the ovary, PhCRT mRNA was most abundant after pollination and reached maximum at the late stage of the progamic phase, when pollen tubes grow into the ovules and fertilization occurs. PhCRT mRNA transcripts were seen to accumulate predominantly in transmitting tract cells of maturing and receptive stigma, in germinated pollen/growing tubes, and at the micropylar region of the ovule, where the female gametophyte is located. From these results, we suggest that PhCRT gene expression is up-regulated during secretory activity of the pistil transmitting tract cells, pollen germination and outgrowth of the tubes, and then during gamete fusion and early embryogenesis.

Disruption of ionic interactions between the nucleotide binding domain 1 (NBD1) and middle (M) domain in Hsp100 disaggregase unleashes toxic hyperactivity and partial independence from Hsp70.

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.

[The multifunctionality of CHIP protein in the protein quality-control system]. / Wielofunkcyjnosc bialka CHIP w systemie kontroli jakosci bialek.

Selective protein degradation depends on their quality being controlled by the cellular system, which includes chaperones involved in protein folding and two degradation systems, the proteasomal and lysosomal. CHIP (carboxyl terminus of Hsp70-interacting protein), an E3 ubiquitin ligase and co-chaperone, serves as a chaperone-degradation system interface. This article reviews the molecular characteristics of CHIP protein, the mechanism of its action, and its role in cellular metabolism and discusses how CHIP dysfunction may lead to neurodegenerative diseases.