Proteomic discovery of cellular substrates of the ClpXP protease reveals five classes of ClpX-recognition signals.
ClpXP is a protease involved in DNA damage repair, stationary-phase gene expression, and ssrA-mediated protein quality control. To date, however, only a handful of ClpXP substrates have been identified. Using a tagged and inactive variant of ClpP, substrates of E. coli ClpXP were trapped in vivo, purified, and identified by mass spectrometry. The more than 50 trapped proteins include transcription factors, metabolic enzymes, and proteins involved in the starvation and oxidative stress responses. Analysis of the sequences of the trapped proteins revealed five recurring motifs: two located at the C terminus of proteins, and three N-terminal motifs. Deletion analysis, fusion proteins, and point mutations established that sequences from each motif class targeted proteins for degradation by ClpXP. These results represent a description of general rules governing substrate recognition by a AAA+ family ATPase and suggest strategies for regulation of protein degradation.
Flynn
Julia M
JM
Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
Neher
Saskia B
SB
Kim
Yong In
YI
Sauer
Robert T
RT
Baker
Tania A
TA
D000074183:ATPases Associated with Diverse Cellular Activities; D000251:Adenosine Triphosphatases / Q000737:chemistry* / Q000378:metabolism*; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D015153:Blotting, Western; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D049071:Endopeptidase Clp; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins; D017353:Gene Deletion; D013058:Mass Spectrometry; D018832:Molecular Chaperones; D008969:Molecular Sequence Data; D018384:Oxidative Stress; D010455:Peptides / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D012697:Serine Endopeptidases / Q000737:chemistry* / Q000378:metabolism*; D013997:Time Factors
D029968:Escherichia coli Proteins; D018832:Molecular Chaperones; D010455:Peptides; D011993:Recombinant Fusion Proteins; D012697:Serine Endopeptidases; C483573:ClpXP protease, E coli; D049071:Endopeptidase Clp; D000251:Adenosine Triphosphatases; C088072:ClpX protein, E coli; D000074183:ATPases Associated with Diverse Cellular Activities