Self-processing of FtsH and its implication for the cleavage specificity of this protease.
FtsH, a membrane-bound and ATP-dependent protease of Escherichia coli, is involved in degradation of some of uncomplexed integral membrane proteins and short-lived cytoplasmic proteins. It is composed of an N-terminal membrane-spanning region and a following large cytoplasmic domain that contains ATPase and protease active sites. In the present study, it was found that FtsH undergoes C-terminal processing in vivo. The processing was blocked by loss of function mutations of FtsH. Purified FtsH-His(6)-Myc, a C-terminally tagged derivative of FtsH, was self-processed in vitro. This in vitro processing was observed only in the presence of ATP and not in the presence of adenosine 5'-(beta,gamma-imino)triphosphate (AMP-PNP). Moreover, such processing did not occur in the case of the ATPase motif mutant protein. These results indicated that this processing is a self-catalyzed reaction that needs ATP hydrolysis. Mutations in the hflKC genes that encode a possible modulator of FtsH, and the growth phase of the cells as well, affected the processing. Complementation experiments with genetically constructed variants suggested that both the processed and the unprocessed forms of FtsH are functional. The cleavage was found to occur between Met-640 and Ser-641, removing a heptapeptide from the C-terminus of FtsH. Systematic mutational analyses of Met-640 and Ser-641 revealed preferences for positively charged and hydrophobic amino acid residues at these positions for processing. This cleavage specificity may be shared by the self-cleavage and the substrate-cleavage reactions of this protease.
Akiyama
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Institute for Virus Research, Kyoto University, Kyoto 606-8507, Japan. yakiyama@virus.kyoto-u.ac.jp
D049069:ATP-Dependent Proteases; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D001483:Base Sequence; D002384:Catalysis; D017931:DNA Primers; D029968:Escherichia coli Proteins; D006868:Hydrolysis; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity
D001426:Bacterial Proteins; D017931:DNA Primers; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D011994:Recombinant Proteins; D049069:ATP-Dependent Proteases; C513383:FtsH protein, E coli