Structure and function of Hip, an attenuator of the Hsp70 chaperone cycle

Li, Zhuo; Hartl, F. Ulrich; Bracher, A.

doi:10.1038/nsmb.2608

Cited by 61 publications

(59 citation statements)

References 190 publications

(282 reference statements)

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“…4d, orange versus blue dots). These results suggest that the TPR domain of Sec72 interacts in a similar way as Hip with the ATPase domain of Hsp70 (32). Indeed, a model can be built on the basis of the crystal structure of the Hip-Hsp70 ATPase domain complex, in which the TPR domain of Sec72 replaces that of Hip (Fig.…”

Section: Ssb1 Interaction With the Tpr Domain Of Sec72mentioning

confidence: 91%

“…This interaction is mediated by the ATPase domain of Ssb1 (Fig. 4b), an interaction that is reminiscent of the binding of the TPR domain of Hip to the ATPase domain of Hsp70 (32). Because Hip inhibits the nucleotide exchange of the ATPase domain, we tested whether the TPR domain of Sec72 has a similar activity.…”

Section: Ssb1 Interaction With the Tpr Domain Of Sec72mentioning

confidence: 99%

“…For Ssa1 and Ssb1, the proteins were treated with EDTA overnight to strip off nucleotide prior to anion exchange (MonoQ, GE Healthcare) and size exclusion (S200, GE Healthcare) chromatography (38). Ssb1_ATPase domain (1-384) was purified as described for the ATPase domain of Hsp70 (32). Purified proteins were flash frozen in liquid nitrogen and stored at Ϫ80°C.…”

Section: Sec71-sec72 and Hsp70s In Post-translational Protein Translomentioning

confidence: 99%

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Two alternative binding mechanisms connect the protein translocation Sec71-Sec72 complex with heat shock proteins

Tripathi

Mandon

Gilmore

et al. 2017

Journal of Biological Chemistry

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The biosynthesis of many eukaryotic proteins requires accurate targeting to and translocation across the endoplasmic reticulum membrane. Post-translational protein translocation in yeast requires both the Sec61 translocation channel, and a complex of four additional proteins: Sec63, Sec62, Sec71, and Sec72. The structure and function of these proteins are largely unknown. This pathway also requires the cytosolic Hsp70 protein Ssa1, but whether Ssa1 associates with the translocation machinery to target protein substrates to the membrane is unclear. Here, we use a combined structural and biochemical approach to explore the role of Sec71-Sec72 subcomplex in post-translational protein translocation. To this end, we report a crystal structure of the Sec71-Sec72 complex, which revealed that Sec72 contains a tetratricopeptide repeat (TPR) domain that is anchored to the endoplasmic reticulum membrane by Sec71. We also determined the crystal structure of this TPR domain with a C-terminal peptide derived from Ssa1, which suggests how Sec72 interacts with full-length Ssa1. Surprisingly, Ssb1, a cytoplasmic Hsp70 that binds ribosome-associated nascent polypeptide chains, also binds to the TPR domain of Sec72, even though it lacks the TPR-binding C-terminal residues of Ssa1. We demonstrate that Ssb1 binds through its ATPase domain to the TPR domain, an interaction that leads to inhibition of nucleotide exchange. Taken together, our results suggest that translocation substrates can be recruited to the Sec71-Sec72 complex either post-translationally through Ssa1 or cotranslationally through Ssb1.

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Section: Ssb1 Interaction With the Tpr Domain Of Sec72mentioning

confidence: 91%

Section: Ssb1 Interaction With the Tpr Domain Of Sec72mentioning

confidence: 99%

Section: Sec71-sec72 and Hsp70s In Post-translational Protein Translomentioning

confidence: 99%

See 1 more Smart Citation

Two alternative binding mechanisms connect the protein translocation Sec71-Sec72 complex with heat shock proteins

Tripathi

Mandon

Gilmore

et al. 2017

Journal of Biological Chemistry

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“…However, recent studies suggested that some TPR domain-containing cochaperones interact with sites on Hsp70 or Hsp90 other than the C-terminal motifs (Alvira et al, 2014; Li et al, 2013; Schmid et al, 2012). We therefore sought a more detailed understanding of how CHIP targets chaperone-bound substrates by characterizing its interaction with Hsp70/Hsc70.…”

Section: Introductionmentioning

confidence: 99%

A Bipartite Interaction between Hsp70 and CHIP Regulates Ubiquitination of Chaperoned Client Proteins

et al. 2015

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Summary The ubiquitin ligase CHIP plays an important role in cytosolic protein quality control by ubiquitinating proteins chaperoned by Hsp70/Hsc70 and Hsp90, thereby targeting such substrate proteins for degradation. We present a 2.91 Å resolution structure of the TPR domain of CHIP in complex with the α-helical “lid” subdomain and unstructured “tail” of Hsc70. Surprisingly, the CHIP-TPR interacts with determinants within both the Hsc70-lid subdomain and the C-terminal PTIEEVD motif of the tail, exhibiting a novel mode of interaction between chaperones and TPR domains. We demonstrate that the interaction between CHIP and the Hsc70-lid subdomain is required for proper ubiquitination of Hsp70/Hsc70 or Hsp70/Hsc70-bound substrate proteins. Post-translational modifications of the Hsc70 lid and tail disrupt key contacts with the CHIP-TPR and may regulate CHIP-mediated ubiquitination. Our study shows how CHIP docks onto Hsp70/Hsc70 and defines a new bipartite mode of interaction between TPR domains and their binding partners.

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“…A transient association is optimal in the cellular context, as it prevents the nonproductive hydrolysis of ATP in the absence of client proteins. This model has also been verified for the Hsp70 folding cycle in yeast and mammalian cells (66,67). Furthermore, optimal refolding activity occurs at substoichiometric levels of J proteins in different experimental systems (68)(69)(70).…”

Section: Discussionmentioning

confidence: 65%

Chaperone-Assisted Protein Folding Is Critical for Yellow Fever Virus NS3/4A Cleavage and Replication

Bozzacco

Andréo

et al. 2016

J Virol

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DNAJC14, a heat shock protein 40 (Hsp40) cochaperone, assists with Hsp70-mediated protein folding. Overexpressed DNAJC14 is targeted to sites of yellow fever virus (YFV) replication complex (RC) formation, where it interacts with viral nonstructural (NS) proteins and inhibits viral RNA replication. How RCs are assembled and the roles of chaperones in this coordinated process are largely unknown. We hypothesized that chaperones are diverted from their normal cellular protein quality control function to play similar roles during viral infection. Here, we show that DNAJC14 overexpression affects YFV polyprotein processing and alters RC assembly. We monitored YFV NS2A-5 polyprotein processing by the viral NS2B-3 protease in DNAJC14-overexpressing cells. Notably, DNAJC14 mutants that did not inhibit YFV replication had minimal effects on polyprotein processing, while overexpressed wild-type DNAJC14 affected the NS3/4A and NS4A/2K cleavage sites, resulting in altered NS3-to-NS3-4A ratios. This suggests that DNAJC14's folding activity normally modulates NS3/4A/2K cleavage events to liberate appropriate levels of NS3 and NS4A and promote RC formation. We introduced amino acid substitutions at the NS3/4A site to alter the levels of the NS3 and NS4A products and examined their effects on YFV replication. Residues with reduced cleavage efficiency did not support viral RNA replication, and only revertant viruses with a restored wild-type arginine or lysine residue at the NS3/4A site were obtained. We conclude that DNAJC14 inhibition of RC formation upon DNAJC14 overexpression is likely due to chaperone dysregulation and that YFV probably utilizes DNAJC14's cochaperone function to modulate processing at the NS3/4A site as a mechanism ensuring virus replication. IMPORTANCEFlaviviruses are single-stranded RNA viruses that cause a wide range of illnesses. Upon host cell entry, the viral genome is translated on endoplasmic reticulum (ER) membranes to produce a single polyprotein, which is cleaved by host and viral proteases to generate viral proteins required for genome replication and virion production. Several studies suggest a role for molecular chaperones during these processes. While the details of chaperone roles have been elusive, in this report we show that overexpression of the ER-resident cochaperone DNAJC14 affects YFV polyprotein processing at the NS3/4A site. This work reveals that DNAJC14 modulation of NS3/4A site processing is an important mechanism to ensure virus replication. Our work highlights the importance of finely regulating flavivirus polyprotein processing. In addition, it suggests future studies to address similarities and/or differences among flaviviruses and to interrogate the precise mechanisms employed for polyprotein processing, a critical step that can ultimately be targeted for novel drug development.

show abstract

Structure and function of Hip, an attenuator of the Hsp70 chaperone cycle

Cited by 61 publications

References 190 publications

Two alternative binding mechanisms connect the protein translocation Sec71-Sec72 complex with heat shock proteins

Two alternative binding mechanisms connect the protein translocation Sec71-Sec72 complex with heat shock proteins

A Bipartite Interaction between Hsp70 and CHIP Regulates Ubiquitination of Chaperoned Client Proteins

Chaperone-Assisted Protein Folding Is Critical for Yellow Fever Virus NS3/4A Cleavage and Replication

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