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Reduction in adaptor amounts establishes degradation hierarchy among protease substrates.

ABSTRACT: ATP-dependent proteases control critical cellular processes, including development, physiology, and virulence. A given protease may recognize a substrate directly via an unfoldase domain or subunit or indirectly via an adaptor that delivers the substrate to the unfoldase. We now report that cells achieve differential stability among substrates of a given protease by modulating adaptor amounts. We establish that the regulatory protein PhoP represses transcription of the gene specifying the ClpAP protease adaptor ClpS when the bacteria Salmonella enterica and Escherichia coli experience low cytoplasmic Mg2+ The resulting decrease in ClpS amounts diminishes proteolysis of several ClpSAP-dependent substrates, including the putrescine aminotransferase Oat, which heightens the formation of antibiotic persisters, and the transcriptional regulators UvrY and PhoP, which alter the expression of genes controlled by these proteins. By contrast, the decrease in ClpS amounts did not impact the abundance of the ClpSAP substrate FtsA, reflecting that FtsA binds to ClpS more tightly than to UvrY and PhoP. Our findings show how physiological conditions that reduce adaptor amounts modify the abundance of selected substrates of a given protease.

SUBMITTER: Yeom J

PROVIDER: S-EPMC5948988 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:In Escherichia coli, ClpYQ (HslUV) is a two-component ATP-dependent protease composed of ClpY (HslU), an ATPase with unfolding activity, and ClpQ (HslV), a peptidase. In the ClpYQ proteolytic complex, the hexameric rings of ClpY (HslU) are responsible for protein recognition, unfolding, and translocation into the proteolytic inner chamber of the dodecameric ClpQ (HslV). Each of the three domains, N, I, and C, in ClpY has its own distinct activity. The double loops (amino acids [aa] 137 to 150 and 175 to 209) in domain I of ClpY are necessary for initial recognition/tethering of natural substrates such as SulA, a cell division inhibitor protein. The highly conserved sequence GYVG (aa 90 to 93) pore I site, along with the GESSG pore II site (aa 265 to 269), contribute to the central pore of ClpY in domain N. These two central loops of ClpY are in the center of its hexameric ring in which the energy of ATP hydrolysis allows substrate translocation and then degradation by ClpQ. However, no data have been obtained to determine the effect of the central loops on substrate binding or as part of the processivity of the ClpYQ complex. Thus, we probed the features of ClpY important for substrate engagement and protease processivity via random PCR or site-specific mutagenesis. In yeast two-hybrid analysis and pulldown assays, using isolated ClpY mutants and the pore I or pore II site of ClpY, each was examined for its influence on the adjoining structural regions of the substrates. The pore I site is essential for the translocation of the engaged substrates. Our in vivo study of the ClpY mutants also revealed that an ATP-binding site in domain N, separate from its role in polypeptide (ClpY) oligomerization, is required for complex formation with ClpQ. Additionally, we found that the tyrosine residue at position 408 in ClpY is critical for stabilization of hexamer formation between subunits. Therefore, our studies suggest that stepwise activities of the ClpYQ protease are necessary to facilitate the processive degradation of its natural substrates.

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Reduction in adaptor amounts establishes degradation hierarchy among protease substrates.

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