scholarly journals Pathway of Hsp70 interactions at the ribosome

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Kanghyun Lee ◽  
Thomas Ziegelhoffer ◽  
Wojciech Delewski ◽  
Scott E. Berger ◽  
Grzegorz Sabat ◽  
...  
Keyword(s):  
Atpase Activity ◽  
Molecular Chaperone ◽  
Ribosomal Proteins ◽  
Atp Hydrolysis ◽  
Site Specific ◽  
Nascent Chain ◽  
J Domain ◽  
Domain Protein ◽  
Chain Interaction

AbstractIn eukaryotes, an Hsp70 molecular chaperone triad assists folding of nascent chains emerging from the ribosome tunnel. In fungi, the triad consists of canonical Hsp70 Ssb, atypical Hsp70 Ssz1 and J-domain protein cochaperone Zuo1. Zuo1 binds the ribosome at the tunnel exit. Zuo1 also binds Ssz1, tethering it to the ribosome, while its J-domain stimulates Ssb’s ATPase activity to drive efficient nascent chain interaction. But the function of Ssz1 and how Ssb engages at the ribosome are not well understood. Employing in vivo site-specific crosslinking, we found that Ssb(ATP) heterodimerizes with Ssz1. Ssb, in a manner consistent with the ADP conformation, also crosslinks to ribosomal proteins across the tunnel exit from Zuo1. These two modes of Hsp70 Ssb interaction at the ribosome suggest a functionally efficient interaction pathway: first, Ssb(ATP) with Ssz1, allowing optimal J-domain and nascent chain engagement; then, after ATP hydrolysis, Ssb(ADP) directly with the ribosome.

scholarly journals Specific Molecular Chaperone Interactions and an ATP-dependent Conformational Change Are Required during Posttranslational Protein Translocation into the Yeast ER

1998 ◽  
Vol 9 (12) ◽  
pp. 3533-3545 ◽  
Author(s):  
Amie J. McClellan ◽  
James B. Endres ◽  
Joseph P. Vogel ◽  
Debra Palazzi ◽  
Mark D. Rose ◽  
...  
Keyword(s):  
Atpase Activity ◽  
Conformational Change ◽  
Molecular Chaperone ◽  
Atp Hydrolysis ◽  
Protein Translocation ◽  
Wild Type ◽  
J Domain ◽  
Chaperone Interactions ◽  
Dependent Interaction ◽  
Er Membrane

The posttranslational translocation of proteins across the endoplasmic reticulum (ER) membrane in yeast requires ATP hydrolysis and the action of hsc70s (DnaK homologues) and DnaJ homologues in both the cytosol and ER lumen. Although the cytosolic hsc70 (Ssa1p) and the ER lumenal hsc70 (BiP) are homologous, they cannot substitute for one another, possibly because they interact with specific DnaJ homologues on each side of the ER membrane. To investigate this possibility, we purified Ssa1p, BiP, Ydj1p (a cytosolic DnaJ homologue), and a GST–63Jp fusion protein containing the lumenal DnaJ region of Sec63p. We observed that BiP, but not Ssa1p, is able to associate with GST–63Jp and that Ydj1p stimulates the ATPase activity of Ssa1p up to 10-fold but increases the ATPase activity of BiP by <2-fold. In addition, Ydj1p and ATP trigger the release of an unfolded polypeptide from Ssa1p but not from BiP. To understand further how BiP drives protein translocation, we purified four dominant lethal mutants of BiP. We discovered that each mutant is defective for ATP hydrolysis, fails to undergo an ATP-dependent conformational change, and cannot interact with GST–63Jp. Measurements of protein translocation into reconstituted proteoliposomes indicate that the mutants inhibit translocation even in the presence of wild-type BiP. We conclude that a conformation- and ATP-dependent interaction of BiP with the J domain of Sec63p is essential for protein translocation and that the specificity of hsc70 action is dictated by their DnaJ partners.

scholarly journals Dissection of Swa2p/Auxilin Domain Requirements for Cochaperoning Hsp70 Clathrin-uncoating Activity In Vivo

2006 ◽  
Vol 17 (7) ◽  
pp. 3281-3290 ◽  
Author(s):  
Jing Xiao ◽  
Leslie S. Kim ◽  
Todd R. Graham
Keyword(s):  
Atpase Activity ◽  
Point Mutation ◽  
Mutational Analysis ◽  
Weak Interactions ◽  
Tetratricopeptide Repeat ◽  
Uba Domain ◽  
J Domain ◽  
Tpr Domain

The auxilin family of J-domain proteins load Hsp70 onto clathrin-coated vesicles (CCVs) to drive uncoating. In vitro, auxilin function requires its ability to bind clathrin and stimulate Hsp70 ATPase activity via its J-domain. To test these requirements in vivo, we performed a mutational analysis of Swa2p, the yeast auxilin ortholog. Swa2p is a modular protein with three N-terminal clathrin-binding (CB) motifs, a ubiquitin association (UBA) domain, a tetratricopeptide repeat (TPR) domain, and a C-terminal J-domain. In vitro, clathrin binding is mediated by multiple weak interactions, but a Swa2p truncation lacking two CB motifs and the UBA domain retains nearly full function in vivo. Deletion of all CB motifs strongly abrogates clathrin disassembly but does not eliminate Swa2p function in vivo. Surprisingly, mutation of the invariant HPD motif within the J-domain to AAA only partially affects Swa2p function. Similarly, a TPR point mutation (G388R) causes a modest phenotype. However, Swa2p function is abolished when these TPR and J mutations are combined. The TPR and J-domains are not functionally redundant because deletion of either domain renders Swa2p nonfunctional. These data suggest that the TPR and J-domains collaborate in a bipartite interaction with Hsp70 to regulate its activity in clathrin disassembly.

Caenorhabditis elegans auxilin: a J-domain protein essential for clathrin-mediated endocytosis in vivo

2001 ◽  
Vol 3 (2) ◽  
pp. 215-219 ◽  
Author(s):  
Tsvika Greener ◽  
Barth Grant ◽  
Yinhua Zhang ◽  
Xufeng Wu ◽  
Lois E. Greene ◽  
...  
Keyword(s):  
Caenorhabditis Elegans ◽  
J Domain ◽  
Domain Protein

scholarly journals Biochemical Convergence of Mitochondrial Hsp70 System Specialized in Iron–Sulfur Cluster Biogenesis

2020 ◽  
Vol 21 (9) ◽  
pp. 3326 ◽  
Author(s):  
Malgorzata Kleczewska ◽  
Aneta Grabinska ◽  
Marcin Jelen ◽  
Milena Stolarska ◽  
Brenda Schilke ◽  
...  
Keyword(s):  
Atpase Activity ◽  
Biochemical Properties ◽  
Iron Sulfur Cluster ◽  
Sulfur Cluster ◽  
Iron Sulfur ◽  
Hsp70 Chaperone ◽  
J Domain ◽  
Domain Protein ◽  
Mitochondrial Hsp70 ◽  
Practical Implications

Mitochondria play a central role in the biogenesis of iron–sulfur cluster(s) (FeS), protein cofactors needed for many cellular activities. After assembly on scaffold protein Isu, the cluster is transferred onto a recipient apo-protein. Transfer requires Isu interaction with an Hsp70 chaperone system that includes a dedicated J-domain protein co-chaperone (Hsc20). Hsc20 stimulates Hsp70′s ATPase activity, thus stabilizing the critical Isu–Hsp70 interaction. While most eukaryotes utilize a multifunctional mitochondrial (mt)Hsp70, yeast employ another Hsp70 (Ssq1), a product of mtHsp70 gene duplication. Ssq1 became specialized in FeS biogenesis, recapitulating the process in bacteria, where specialized Hsp70 HscA cooperates exclusively with an ortholog of Hsc20. While it is well established that Ssq1 and HscA converged functionally for FeS transfer, whether these two Hsp70s possess similar biochemical properties was not known. Here, we show that overall HscA and Ssq1 biochemical properties are very similar, despite subtle differences being apparent - the ATPase activity of HscA is stimulated to a somewhat higher levels by Isu and Hsc20, while Ssq1 has a higher affinity for Isu and for Hsc20. HscA/Ssq1 are a unique example of biochemical convergence of distantly related Hsp70s, with practical implications, crossover experimental results can be combined, facilitating understanding of the FeS transfer process.

scholarly journals The Walker B motif of the second nucleotide-binding domain (NBD2) of CFTR plays a key role in ATPase activity by the NBD1–NBD2 heterodimer

2006 ◽  
Vol 401 (2) ◽  
pp. 581-586 ◽  
Author(s):  
Fiona L. L. Stratford ◽  
Mohabir Ramjeesingh ◽  
Joanne C. Cheung ◽  
Ling-JUN Huan ◽  
Christine E. Bear
Keyword(s):  
Atpase Activity ◽  
Atp Hydrolysis ◽  
Nucleotide Binding ◽  
Vital Role ◽  
Atp Binding ◽  
Primary Role ◽  
Binding Domains ◽  
Membrane Spanning ◽  
Atp Binding And Hydrolysis

CFTR (cystic fibrosis transmembrane conductance regulator), a member of the ABC (ATP-binding cassette) superfamily of membrane proteins, possesses two NBDs (nucleotide-binding domains) in addition to two MSDs (membrane spanning domains) and the regulatory ‘R’ domain. The two NBDs of CFTR have been modelled as a heterodimer, stabilized by ATP binding at two sites in the NBD interface. It has been suggested that ATP hydrolysis occurs at only one of these sites as the putative catalytic base is only conserved in NBD2 of CFTR (Glu1371), but not in NBD1 where the corresponding residue is a serine, Ser573. Previously, we showed that fragments of CFTR corresponding to NBD1 and NBD2 can be purified and co-reconstituted to form a heterodimer capable of ATPase activity. In the present study, we show that the two NBD fragments form a complex in vivo, supporting the utility of this model system to evaluate the role of Glu1371 in ATP binding and hydrolysis. The present studies revealed that a mutant NBD2 (E1371Q) retains wild-type nucleotide binding affinity of NBD2. On the other hand, this substitution abolished the ATPase activity formed by the co-purified complex. Interestingly, introduction of a glutamate residue in place of the non-conserved Ser573 in NBD1 did not confer additional ATPase activity by the heterodimer, implicating a vital role for multiple residues in formation of the catalytic site. These findings provide the first biochemical evidence suggesting that the Walker B residue: Glu1371, plays a primary role in the ATPase activity conferred by the NBD1–NBD2 heterodimer.

scholarly journals Biological and Structural Basis for Aha1 Regulation of Hsp90 ATPase Activity in Maintaining Proteostasis in the Human Disease Cystic Fibrosis

2010 ◽  
Vol 21 (6) ◽  
pp. 871-884 ◽  
Author(s):  
Atanas V. Koulov ◽  
Paul LaPointe ◽  
Bingwen Lu ◽  
Abbas Razvi ◽  
Judith Coppinger ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Atpase Activity ◽  
Protein Misfolding ◽  
Atp Hydrolysis ◽  
Structural Basis ◽  
Inherited Disease ◽  
Hsp90 Chaperone ◽  
Terminal Domains

The activator of Hsp90 ATPase 1, Aha1, has been shown to participate in the Hsp90 chaperone cycle by stimulating the low intrinsic ATPase activity of Hsp90. To elucidate the structural basis for ATPase stimulation of human Hsp90 by human Aha1, we have developed novel mass spectrometry approaches that demonstrate that the N- and C-terminal domains of Aha1 cooperatively bind across the dimer interface of Hsp90 to modulate the ATP hydrolysis cycle and client activity in vivo. Mutations in both the N- and C-terminal domains of Aha1 impair its ability to bind Hsp90 and stimulate its ATPase activity in vitro and impair in vivo the ability of the Hsp90 system to modulate the folding and trafficking of wild-type and variant (ΔF508) cystic fibrosis transmembrane conductance regulator (CFTR) responsible for the inherited disease cystic fibrosis (CF). We now propose a general model for the role of Aha1 in the Hsp90 ATPase cycle in proteostasis whereby Aha1 regulates the dwell time of Hsp90 with client. We suggest that Aha1 activity integrates chaperone function with client folding energetics by modulating ATPase sensitive N-terminal dimer structural transitions, thereby protecting transient folding intermediates in vivo that could contribute to protein misfolding systems disorders such as CF when destabilized.

scholarly journals Cdc6 ATPase activity disengages Cdc6 from the pre-replicative complex to promote DNA replication

eLife ◽  
2015 ◽  
Vol 4 ◽  
Author(s):  
FuJung Chang ◽  
Alberto Riera ◽  
Cecile Evrin ◽  
Jingchuan Sun ◽  
Huilin Li ◽  
...  
Keyword(s):  
Dna Replication ◽  
Atpase Activity ◽  
Atp Hydrolysis ◽  
S Phase ◽  
G1 Phase ◽  
Helicase Loading ◽  
Mcm Helicase

To initiate DNA replication, cells first load an MCM helicase double hexamer at origins in a reaction requiring ORC, Cdc6, and Cdt1, also called pre-replicative complex (pre-RC) assembly. The essential mechanistic role of Cdc6 ATP hydrolysis in this reaction is still incompletely understood. Here, we show that although Cdc6 ATP hydrolysis is essential to initiate DNA replication, it is not essential for MCM loading. Using purified proteins, an ATPase-defective Cdc6 mutant ‘Cdc6-E224Q’ promoted MCM loading on DNA. Cdc6-E224Q also promoted MCM binding at origins in vivo but cells remained blocked in G1-phase. If after loading MCM, Cdc6-E224Q was degraded, cells entered an apparently normal S-phase and replicated DNA, a phenotype seen with two additional Cdc6 ATPase-defective mutants. Cdc6 ATP hydrolysis is therefore required for Cdc6 disengagement from the pre-RC after helicase loading to advance subsequent steps in helicase activation in vivo.

scholarly journals Prefabrication of a ribosomal protein subcomplex essential for eukaryotic ribosome formation

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Cohue Peña ◽  
Sabina Schütz ◽  
Ute Fischer ◽  
Yiming Chang ◽  
Vikram G Panse
Keyword(s):  
Atpase Activity ◽  
Structural Feature ◽  
Ribosomal Protein ◽  
Ribosomal Proteins ◽  
Spatial Clustering ◽  
Functional Importance ◽  
Eukaryotic Ribosome ◽  
Ribosome Formation

Spatial clustering of ribosomal proteins (r-proteins) through tertiary interactions is a striking structural feature of the eukaryotic ribosome. However, the functional importance of these intricate inter-connections, and how they are established is currently unclear. Here, we reveal that a conserved ATPase, Fap7, organizes interactions between neighboring r-proteins uS11 and eS26 prior to their delivery to the earliest ribosome precursor, the 90S. In vitro, uS11 only when bound to Fap7 becomes competent to recruit eS26 through tertiary contacts found between these r-proteins on the mature ribosome. Subsequently, Fap7 ATPase activity unloads the uS11:eS26 subcomplex onto its rRNA binding site, and therefore ensures stoichiometric integration of these r-proteins into the 90S. Fap7-depletion in vivo renders uS11 susceptible to proteolysis, and precludes eS26 incorporation into the 90S. Thus, prefabrication of a native-like r-protein subcomplex drives efficient and accurate construction of the eukaryotic ribosome.

scholarly journals Nitration of specific tyrosines in FoF1 ATP synthase and activity loss in aging

2010 ◽  
Vol 298 (5) ◽  
pp. E978-E987 ◽  
Author(s):  
Virginia Haynes ◽  
Nathaniel J. Traaseth ◽  
Sarah Elfering ◽  
Yasuko Fujisawa ◽  
Cecilia Giulivi
Keyword(s):  
Atpase Activity ◽  
Atp Hydrolysis ◽  
Solvent Accessibility ◽  
Consensus Sequence ◽  
Β Subunit ◽  
Nitrative Stress ◽  
Old Rats ◽  
Activity Loss ◽  
Major Target

It has been reported that C-nitration of proteins occurs under nitrative/oxidative stress; however, its role in pathophysiological situations is not fully understood. In this study, we determined that nitration of Tyr345 and Tyr368 in the β-subunit of the mitochondrial FoF1-ATPase is a major target for nitrative stress in rat liver under in vivo conditions. The chemical characteristics of these Tyr make them suitable for a facilitated nitration (solvent accessibility, consensus sequence, and p Ka). Moreover, β-subunit nitration increased significantly with the age of the rats (from 4 to 80 weeks old) and correlated with decreased ATP hydrolysis and synthesis rates. Although its affinity for ATP binding was unchanged, maximal ATPase activity decreased between young and old rats by a factor of two. These changes directly impacted the available ATP concentration in vivo, and it was expected that they would affect multiple cellular ATP-dependent processes. For instance, at least 50% of available [ATP] in the liver of older rats would have to be committed to sustain maximal Na+-K+-ATPase activity, whereas only 30% would be required for young rats. If this requirement was not fulfilled, the osmoregulation and Na+-nutrient cotransport in liver of older rats would be compromised. On the basis of our studies, we propose that targeted nitration of the β-subunit is an early marker for nitrative stress and aging.

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