The Clp Proteases ofBacillus subtilisAre Directly Involved in Degradation of Misfolded Proteins

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Abstract
The presence of the heat stress response-related ATPases ClpC and ClpX or the peptidase ClpP in the cell is crucial for tolerance of many forms of stress inBacillus subtilis. Assays for detection of defects in protein degradation suggest that ClpC, ClpP, and ClpX participate directly in overall proteolysis of misfolded proteins. Turnover rates for abnormal puromycyl peptides are significantly decreased inclpC,clpP, andclpXmutant cells. Electron-dense aggregates, most likely due to the accumulation of misfolded proteins, were noticed in studies of ultrathin cryosections inclpCandclpPmutant cells even under nonstress conditions. In contrast, in the wild type orclpXmutants such aggregates could only be observed after heat shock. This phenomenon supports the assumption thatclpCandclpPmutants are deficient in the ability to solubilize or degrade damaged and aggregated proteins, the accumulation of which is toxic for the cell. By using immunogold labeling with antibodies raised against ClpC, ClpP, and ClpX, the Clp proteins were localized in these aggregates, showing that the Clp proteins act at this level in vivo.