Assistance for a Chaperone

Willmund, Felix; Hinnenberger, Manuela; Nick, Sabine; Schulz-Raffelt, Miriam; Mühlhaus, Timo; Schroda, Michael

doi:10.1074/jbc.m708431200

Cited by 25 publications

(20 citation statements)

References 43 publications

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“…When expressed in E. coli, the solubility of Ssc1/mtHsp70 and constructs of mtHsp70 were dependent on co-expression of Hep1 (29,31,32). Similar observations were made for chloroplast Hsp70B and its co-chaperone Hep2 (33).…”

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confidence: 60%

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ATPase Domain and Interdomain Linker Play a Key Role in Aggregation of Mitochondrial Hsp70 Chaperone Ssc1

Blamowska¹,

Sichting²,

Mapa

et al. 2010

Journal of Biological Chemistry

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The co-chaperone Hep1 is required to prevent the aggregation of mitochondrial Hsp70 proteins. We have analyzed the interaction of Hep1 with mitochondrial Hsp70 (Ssc1) and the determinants in Ssc1 that make it prone to aggregation. The ATPase and peptide binding domain (PBD) of Hsp70 proteins are connected by a linker segment that mediates interdomain communication between the domains. We show here that the minimal Hep1 binding entity of Ssc1 consists of the ATPase domain and the interdomain linker. In the absence of Hep1, the ATPase domain with the interdomain linker had the tendency to aggregate, in contrast to the ATPase domain with the mutated linker segment or without linker, and in contrast to the PBD. The closest homolog of Ssc1, bacterial DnaK, and a Ssc1 chimera, in which a segment of the ATPase domain of Ssc1 was replaced by the corresponding segment from DnaK, did not aggregate in ⌬hep1 mitochondria. The propensity to aggregate appears to be a specific property of the mitochondrial Hsp70 proteins. The ATPase domain in combination with the interdomain linker is crucial for aggregation of Ssc1. In conclusion, our results suggest that interdomain communication makes Ssc1 prone to aggregation. Hep1 counteracts aggregation by binding to this aggregation-prone conformer.Hsp70 chaperones mediate important processes in prokaryotes and eukaryotes such as folding, translocation, and prevention of aggregation of proteins (1-3). They are highly conserved in respect to both, amino acid sequence and structure. An N-terminal ATP binding domain (ATPase domain) and a C-terminal peptide binding domain (PBD) 3 are connected by a short hydrophobic interdomain linker. The PBD binds unfolded polypeptides. It acts in cooperation with the ATPase domain, which binds and hydrolyzes ATP. The nucleotide state of the ATPase domain determines the properties of the PBD. In the ATP state the PBD has a low affinity for substrates and binds and releases substrates rapidly. Hydrolysis leads to the ADP bound state of the ATPase domain in which the PBD adopts a closed conformation and binds substrates with high affinity. On the other hand, substrate binding to the PBD stimulates the hydrolysis rate of the ATPase domain. Thus, structural changes in one domain induce conformational alterations in the other domain (4 -10). This interdomain communication is due to changing interdomain contacts. Such contacts have been reported for all nucleotide states (11-15); however, it is not clear whether contacts of Hsp70 proteins observed in the absence of ATP are physiologically relevant (11,13,15). In the ATP bound state the interdomain linker binds to a hydrophobic cleft present in the ATPase domain and stimulates the ATPase activity (15). Together with other studies this suggests an important role of the linker in the mechanism of interdomain communication (5,(15)(16)(17)(18). Mutations within the interdomain linker inhibit the allosteric control of the ATPase domain by the PBD (5, 16, 18). DnaK constructs consisting of the ATPase domain and the in...

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confidence: 60%

“…In addition, human Hep1 may have a stimulatory effect on the catalytic activity of the ATPase domain of mtHsp70. Interestingly, a Hep2 homolog has been identified in chloroplasts in the algae Chlamydomonas reinhardtii (33). This raises intriguing questions on the evolutionary origin of Hep proteins and their functions in the two organelles.…”

Section: Discussionmentioning

confidence: 99%

ATPase Domain and Interdomain Linker Play a Key Role in Aggregation of Mitochondrial Hsp70 Chaperone Ssc1

Blamowska¹,

Sichting²,

Mapa

et al. 2010

Journal of Biological Chemistry

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“…Important especially for mathematical modeling in systems biology is the knowledge of the absolute concentrations of biomolecules in a cell (Pratt et al, 2006). For proteins, this information is difficult to obtain and before the introduction of mass spectrometry was done for example by photospectroscopy on proteins harboring light-absorbing cofactors, by radioligand assays, or by immunoassays requiring protein standards (Merchant et al, 1991; Murakami et al, 1997; Willmund et al, 2008). …”

Section: Introductionmentioning

confidence: 99%

Absolute Quantification of Major Photosynthetic Protein Complexes in Chlamydomonas reinhardtii Using Quantification Concatamers (QconCATs)

et al. 2018

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For modeling approaches in systems biology, knowledge of the absolute abundances of cellular proteins is essential. One way to gain this knowledge is the use of quantification concatamers (QconCATs), which are synthetic proteins consisting of proteotypic peptides derived from the target proteins to be quantified. The QconCAT protein is labeled with a heavy isotope upon expression in E. coli and known amounts of the purified protein are spiked into a whole cell protein extract. Upon tryptic digestion, labeled and unlabeled peptides are released from the QconCAT protein and the native proteins, respectively, and both are quantified by LC-MS/MS. The labeled Q-peptides then serve as standards for determining the absolute quantity of the native peptides/proteins. Here, we have applied the QconCAT approach to Chlamydomonas reinhardtii for the absolute quantification of the major proteins and protein complexes driving photosynthetic light reactions in the thylakoid membranes and carbon fixation in the pyrenoid. We found that with 25.2 attomol/cell the Rubisco large subunit makes up 6.6% of all proteins in a Chlamydomonas cell and with this exceeds the amount of the small subunit by a factor of 1.56. EPYC1, which links Rubisco to form the pyrenoid, is eight times less abundant than RBCS, and Rubisco activase is 32-times less abundant than RBCS. With 5.2 attomol/cell, photosystem II is the most abundant complex involved in the photosynthetic light reactions, followed by plastocyanin, photosystem I and the cytochrome b6/f complex, which range between 2.9 and 3.5 attomol/cell. The least abundant complex is the ATP synthase with 2 attomol/cell. While applying the QconCAT approach, we have been able to identify many potential pitfalls associated with this technique. We analyze and discuss these pitfalls in detail and provide an optimized workflow for future applications of this technique.

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“…Coexpression of Ssc1 with Hep1 in Escherichia coli led to the production of soluble Ssc1, whereas expression of Ssc1 alone yielded insoluble chaperone (23). In addition, the Chlamydomonas reinhardtii chloroplast Hsp70B could only be produced as a soluble functional protein in bacteria when it was coexpressed with Hep2 (24). Currently, the nature of chaperone misfolding reactions and escort protein regulation of chaperone folding are unclear.…”

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confidence: 99%

“…Bacterial expression studies have provided evidence that eukaryotic escort proteins are sufficient to promote the solubility of their cognate hsp70 chaperones (23,24). Coexpression of Ssc1 with Hep1 in Escherichia coli led to the production of soluble Ssc1, whereas expression of Ssc1 alone yielded insoluble chaperone (23).…”

mentioning

confidence: 99%

The Human Escort Protein Hep Binds to the ATPase Domain of Mitochondrial Hsp70 and Regulates ATP Hydrolysis

Zhai

Stanworth

Liu

et al. 2008

Journal of Biological Chemistry

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Hsp70 escort proteins (Hep) have been implicated as essential for maintaining the function of yeast mitochondrial hsp70 molecular chaperones (mtHsp70), but the role that escort proteins play in regulating mammalian chaperone folding and function has not been established. We present evidence that human mtHsp70 exhibits limited solubility due to aggregation mediated by its ATPase domain and show that human Hep directly enhances chaperone solubility through interactions with this domain. In the absence of Hep, mtHsp70 was insoluble when expressed in Escherichia coli, as was its isolated ATPase domain and a chimera having this domain fused to the peptide-binding domain of HscA, a soluble monomeric chaperone. In contrast, these proteins all exhibited increased solubility when expressed in the presence of Hep. In vitro studies further revealed that purified Hep regulates the interaction of mtHsp70 with nucleotides. Full-length mtHsp70 exhibited slow intrinsic ATP hydrolysis activity (6.8 ؎ 0.2 ؋ 10 ؊4 s ؊1 ) at 25°C, which was stimulated up to 49-fold by Hep. Hep also stimulated the activity of the isolated ATPase domain, albeit to a lower maximal extent (11.5-fold). In addition, gel-filtration studies showed that formation of chaperone-escort protein complexes inhibited mtHsp70 self-association, and they revealed that Hep binding to full-length mtHsp70 and its isolated ATPase domain is strongest in the absence of nucleotides. These findings provide evidence that metazoan escort proteins regulate the catalytic activity and solubility of their cognate chaperones, and they indicate that both forms of regulation arise from interactions with the mtHsp70 ATPase domain.

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Assistance for a Chaperone

Cited by 25 publications

References 43 publications

ATPase Domain and Interdomain Linker Play a Key Role in Aggregation of Mitochondrial Hsp70 Chaperone Ssc1

ATPase Domain and Interdomain Linker Play a Key Role in Aggregation of Mitochondrial Hsp70 Chaperone Ssc1

Absolute Quantification of Major Photosynthetic Protein Complexes in Chlamydomonas reinhardtii Using Quantification Concatamers (QconCATs)

The Human Escort Protein Hep Binds to the ATPase Domain of Mitochondrial Hsp70 and Regulates ATP Hydrolysis

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