Evidence for an Unfolding/Threading Mechanism for Protein Disaggregation by Saccharomyces cerevisiae Hsp104

Lum, Ronnie; Tkach, Johnny M.; Vierling, Elizabeth; Glover, John R.

doi:10.1074/jbc.m403777200

Cited by 229 publications

(258 citation statements)

References 40 publications

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“…The prevailing model suggests that the Hsp70 system targets the Hsp104 motor to aggregates (20,22), from which Hsp104 extracts polypeptides using pore loops in D1 and D2, perhaps with assistance from the NTDs (25), by threading the polypeptide through the central channel of the Hsp104 hexamer ring (20,21,26,27). This threading mechanism is similar to that of the structurally homologous ClpA ATPase, which lacks an M-domain and together with ClpP degrades ssrA-tagged proteins (28)(29)(30).…”

mentioning

confidence: 89%

Heat shock protein (Hsp) 70 is an activator of the Hsp104 motor

Lee¹,

Kim²,

Biter³

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

104

111

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Heat shock protein (Hsp) 104 is a ring-forming, protein-remodeling machine that harnesses the energy of ATP binding and hydrolysis to drive protein disaggregation. Although Hsp104 is an active ATPase, the recovery of functional protein requires the speciesspecific cooperation of the Hsp70 system. However, like Hsp104, Hsp70 is an active ATPase, which recognizes aggregated and aggregation-prone proteins, making it difficult to differentiate the mechanistic roles of Hsp104 and Hsp70 during protein disaggregation. Mapping the Hsp70-binding sites in yeast Hsp104 using peptide array technology and photo-cross-linking revealed a striking conservation of the primary Hsp70-binding motifs on the Hsp104 middle-domain across species, despite lack of sequence identity. Remarkably, inserting a Strep-Tactin binding motif at the spatially conserved Hsp70-binding site elicits the Hsp104 protein disaggregating activity that now depends on Strep-Tactin but no longer requires Hsp70/40. Consistent with a Strep-Tactin-dependent activation step, we found that full-length Hsp70 on its own could activate the Hsp104 hexamer by promoting intersubunit coordination, suggesting that Hsp70 is an activator of the Hsp104 motor.ClpB | DnaK | Hsp100 | molecular chaperone

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mentioning

confidence: 89%

Heat shock protein (Hsp) 70 is an activator of the Hsp104 motor

Lee¹,

Kim²,

Biter³

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

104

111

View full text Add to dashboard Cite

show abstract

“…Though the precise mechanism of disaggregation is still elusive, some evidence suggests that Hsp70-Hsp40 remodels protein aggregates and allows the transfer of aggregated polypeptides to the substrate processing pore of Hsp104 ( Figure 10). Hsp104, in an ATP dependent process, then exerts a threading or pulling activity to facilitate extraction of the misfolded polypeptides from the aggregates (Liberek et al, 2008;Lum et al, 2004;Tyedmers et al, 2010). Once inside the Hsp104 cylinder, the polypeptide is disentangled to soluble form.…”

Section: Ii441 Chaperone Mediated Refolding and Disaggregationmentioning

confidence: 99%

Firefly luciferase mutants as sensors of proteome stress

et al. 2011

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“…5 The loops bind polypeptide substrates and are involved in their forced translocation. 11,13 ATP stabilizes the position of the D1 loop, whereas the loop retains its flexibility in the presence of ADP. 14 This observation is consistent with the ATP-induced high-affinity binding of substrates by ClpB and a low affinity of the ADP state 15 and implies a conformational linkage between the channel loop and the ATP-binding site.…”

Section: Introductionmentioning

confidence: 99%

Walker‐A threonine couples nucleotide occupancy with the chaperone activity of the AAA+ ATPase ClpB

Nagy

et al. 2009

Protein Science

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Hexameric AAA1 ATPases induce conformational changes in a variety of macromolecules. AAA1 structures contain the nucleotide-binding P-loop with the Walker A sequence motif: GxxGxGK(T/S). A subfamily of AAA1 sequences contains Asn in the Walker A motif instead of Thr or Ser. This noncanonical subfamily includes torsinA, an ER protein linked to human dystonia and DnaC, a bacterial helicase loader. Role of the noncanonical Walker A motif in the functionality of AAA1 ATPases has not been explored yet. To determine functional effects of introduction of Asn into the Walker A sequence, we replaced the Walker-A Thr with Asn in ClpB, a bacterial AAA1 chaperone which reactivates aggregated proteins. We found that the T-to-N mutation in Walker A partially inhibited the ATPase activity of ClpB, but did not affect the ClpB capability to associate into hexamers. Interestingly, the noncanonical Walker A sequence in ClpB induced preferential binding of ADP vs. ATP and uncoupled the linkage between the ATP-bound conformation and the high-affinity binding to protein aggregates. As a consequence, ClpB with the noncanonical Walker A sequence showed a low chaperone activity in vitro and in vivo. Our results demonstrate a novel role of the Walker-A Thr in sensing the nucleotide's c-phosphate and in maintaining an allosteric linkage between the P-loop and the aggregate binding site of ClpB. We postulate that AAA1 ATPases with the noncanonical Walker A might utilize distinct mechanisms to couple the ATPase cycle with their substrate-remodeling activity.

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Evidence for an Unfolding/Threading Mechanism for Protein Disaggregation by Saccharomyces cerevisiae Hsp104

Abstract: Saccharomyces cerevisiae

Cited by 229 publications

References 40 publications

Heat shock protein (Hsp) 70 is an activator of the Hsp104 motor

Heat shock protein (Hsp) 70 is an activator of the Hsp104 motor

Firefly luciferase mutants as sensors of proteome stress

Walker‐A threonine couples nucleotide occupancy with the chaperone activity of the AAA+ ATPase ClpB

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