Sequence and length recognition of the C-terminal turnover element of LpxC, a soluble substrate of the membrane-bound FtsH protease.
The membrane-anchored FtsH protease is essential in Escherichia coli as it adjusts the cellular amount of LpxC, the key enzyme in lipopolysaccharide (LPS) biosynthesis. Both accumulation and depletion of LpxC are toxic to E. coli. By continuous proteolysis of LpxC, FtsH maintains a low concentration of LpxC and, hence, the proper equilibrium between LPS and phospholipids. The C terminus of LpxC is required for turnover. By adding this tail to glutathione-S-transferase (GST) we show that it is necessary but not sufficient for FtsH-mediated degradation. A detailed mutational analysis revealed six non-polar residues in the C terminus of LpxC that are critical for degradation. Alteration of the C-terminal AVLA motif towards the SsrA-like sequence ALAA directed LpxC to other cellular proteases reinforcing the importance of the C-terminal tail for targeting to FtsH. Short C-terminal truncations stabilized LpxC. Most mutations in the C terminus of LpxC left its enzymatic activity intact as was shown by growth assays, microscopy and 2-keto-3-deoxyoctonate (KDO) determination. The critical length of the turnover element was defined by internal deletions. A C-terminal tail of about 20 amino acids length is required for proteolysis of LpxC by FtsH.
Führer
Frank
F
Lehrstuhl für Biologie der Mikroorganismen, Ruhr-Universität Bochum, Universitätsstrasse 150, NDEF 06/783, 44780, Bochum, Germany.
Müller
Alexandra
A
Baumann
Holger
H
Langklotz
Sina
S
Kutscher
Blanka
B
Narberhaus
Franz
F
D049069:ATP-Dependent Proteases / Q000378:metabolism*; D000581:Amidohydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000378:metabolism*; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D011499:Protein Processing, Post-Translational / Q000235:genetics*; D012995:Solubility; D013329:Structure-Activity Relationship
D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D005982:Glutathione Transferase; D049069:ATP-Dependent Proteases; C513383:FtsH protein, E coli; D000581:Amidohydrolases; C097307:UDP-3-O-acyl-N-acetylglucosamine deacetylase