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E. coli ClpA catalyzed polypeptide translocation is allosterically controlled by the protease ClpP.

ABSTRACT: There are five known ATP-dependent proteases in Escherichia coli (Lon, ClpAP, ClpXP, HslUV, and the membrane-associated FtsH) that catalyze the removal of both misfolded and properly folded proteins in cellular protein quality control pathways. Hexameric ClpA rings associate with one or both faces of the cylindrically shaped tetradecameric ClpP protease. ClpA catalyzes unfolding and translocation of polypeptide substrates into the proteolytic core of ClpP for degradation through repeated cycles of ATP binding and hydrolysis at two nucleotide binding domains on each ClpA monomer. We previously reported a molecular mechanism for ClpA catalyzed polypeptide translocation in the absence of ClpP, including elementary rate constants, overall rate, and the kinetic step size. However, the potential allosteric effect of ClpP on the mechanism of ClpA catalyzed translocation remains unclear. Using single-turnover fluorescence stopped-flow methods, here we report that ClpA, when associated with ClpP, translocates polypeptide with an overall rate of ~35 aa s(-1) and, on average, traverses ~5 aa between two rate-limiting steps with reduced cooperativity between ATP binding sites in the hexameric ring. This is in direct contrast to our previously reported observation that, in the absence of ClpP, ClpA translocates polypeptide substrates with a maximum translocation rate of ~20 aa s(-1) with cooperativity between ATPase sites. Our results demonstrate that ClpP allosterically impacts the polypeptide translocation activity of ClpA by reducing the cooperativity between ATP binding sites.

SUBMITTER: Miller JM

PROVIDER: S-EPMC3918674 | biostudies-literature | 2013 Aug

REPOSITORIES: biostudies-literature

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E. coli ClpA catalyzed polypeptide translocation is allosterically controlled by the protease ClpP.

Miller Justin M JM Miller Justin M JM Lin Jiabei J Li Tao T Lucius Aaron L AL

Journal of molecular biology 20130429 15

There are five known ATP-dependent proteases in Escherichia coli (Lon, ClpAP, ClpXP, HslUV, and the membrane-associated FtsH) that catalyze the removal of both misfolded and properly folded proteins in cellular protein quality control pathways. Hexameric ClpA rings associate with one or both faces of the cylindrically shaped tetradecameric ClpP protease. ClpA catalyzes unfolding and translocation of polypeptide substrates into the proteolytic core of ClpP for degradation through repeated cycles ...[more]

PMID: 23639359

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Project description:The peptidase ClpP is conserved from bacteria to human. In the mitochondrial matrix, it multimerizes and forms a macromolecular proteasome-like cylinder. Because of its known relevance for the mitochondrial unfolded protein response during cell stress, we characterized two ClpP knock-out mouse founder lines and documented similar phenotypes. Ubiquitously, ClpP absence led to accumulation of its interactor protein ClpX without transcript upregulation. Interestingly, most wild-type tissues with substantial ClpP amounts had no detectable ClpX. This inverse correlation suggests that ClpX levels and degradation are regulated by ClpP. The expectation of similar protein levels, in view of a reported association of heptameric ClpP rings with hexameric ClpX rings, was confirmed only in testis of wild-type animals. Germline tissue was exceptional also in its vulnerability to ClpP deletion, with both founder lines showing complete infertility for males and females. Otherwise, ubiquitous mitochondrial dysfunction was apparent from severe growth retardation and reduced spontaneous motor activity of the animals, and from a pronounced decrease in pre-/postnatal survival. Spermatogenesis was found aborted at the spermatid stage, acrosomes and axonemes were not formed. Overall, tissue-specific roles of ClpP were evident by this massive effect for germ cells, mild bioenergetic deficits in muscle and liver tissues, and excellent compensation in brain. ClpX was previously reported to chaperone unfolded proteins and also DNA condensation in mitochondria, so it is likely that this pathway is particularly susceptible in germ cells. In conclusion, our study indicates that the role of ClpP in quality control is indispensable during development for cells with rapid changes of mitochondrial numbers, and is relevant during aging for growth and survival of the organism. Factorial design comparing ClpP knock-out mice with wild type littermates in five different tissues (brain, testis, liver, skeletal and heart muscle)

Dataset Information

E. coli ClpA catalyzed polypeptide translocation is allosterically controlled by the protease ClpP.

Publications

E. coli ClpA catalyzed polypeptide translocation is allosterically controlled by the protease ClpP.

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