title: Two peptide sequences can function cooperatively to facilitate binding and unfolding by ClpA and degradation by ClpAP.
abstract: Clp/Hsp100 proteins comprise a large family of AAA(+) ATPases. Some Clp proteins function alone as molecular chaperones, whereas others act in conjunction with peptidases, forming ATP-dependent proteasome-like compartmentalized proteases. Protein degradation by Clp proteases is regulated primarily by substrate recognition by the Clp ATPase component. The ClpA and ClpX ATPases of Escherichia coli generally recognize short amino acid sequences that are located near the N or C terminus of a substrate. However, both ClpAP and ClpXP are able to degrade proteins in which the end containing the recognition signal is fused to GFP such that the signal is in the interior of the primary sequence of the substrate. Here, we tested whether the internal ClpA recognition signal was the sole element required for targeting the substrate to ClpA. The results show that, in the absence of a high-affinity peptide recognition signal at the terminus, two elements are important for recognition of GFP-RepA fusion proteins by ClpA. One element is the natural ClpA recognition signal located at the junction of GFP and RepA in the fusion protein. The second element is the C-terminal peptide of the fusion protein. Together, these two elements facilitate binding and unfolding by ClpA and degradation by ClpAP. The internal site appears to function similarly to Clp adaptor proteins but, in this case, is covalently attached to the polypeptide containing the terminal tag and both the "adaptor" and "substrate" modules are degraded.
authors.affiliation: Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA.
authors.forename: Joel R
authors.initials: JR
authors.lastname: Hoskins
authors.forename: Sue
authors.initials: S
authors.lastname: Wickner
mesh_terms: D000074183:ATPases Associated with Diverse Cellular Activities; D000251:Adenosine Triphosphatases / Q000737:chemistry; D004247:DNA / Q000737:chemistry; D049071:Endopeptidase Clp / Q000737:chemistry*; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D008954:Models, Biological; D018832:Molecular Chaperones / Q000737:chemistry / Q000378:metabolism; D010455:Peptides / Q000737:chemistry*; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D011489:Protein Denaturation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D013379:Substrate Specificity; D013997:Time Factors
chemical_list: D029968:Escherichia coli Proteins; D018832:Molecular Chaperones; D010455:Peptides; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D004247:DNA; C483568:ClpA protease, E coli; C483572:ClpP protease, E coli; D049071:Endopeptidase Clp; D000251:Adenosine Triphosphatases; C088072:ClpX protein, E coli; D000074183:ATPases Associated with Diverse Cellular Activities