Sequence and Length Recognition of the C-terminal Turnover Element of LpxC, a Soluble Substrate of the Membrane-bound FtsH Protease

Führer, Frank; Müller, Alexandra; Baumann, Holger; Langklotz, Sina; Kutscher, Blanka; Narberhaus, Franz

doi:10.1016/j.jmb.2007.06.083

Cited by 43 publications

(60 citation statements)

References 52 publications

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“…To monitor cellular LPS production of LpxC-expressing WT and FabZ* cells, relative amounts of membrane-bound Kdo were quantified using a Kdo assay derived from Karkhanis et al as described previously (7,22) Microscopic analysis. To analyze the effect of LpxC overexpression in WT and FabZ* cells, 2 l of the induced culture (0.5% arabinose) was fixed in an agarose matrix and inspected with a BX51 microscope (Olympus).…”

Section: Methodsmentioning

confidence: 99%

“…However, it is conceivable that further mechanisms are involved in this critical process. Obviously, not only the relative ratio between PL and LPS but also the quantity of both molecules needs to meet the cellular requirements during different generation times because excessive production of LPS is toxic (6)(7)(8). The regulation of LPS synthesis in Escherichia coli and related enterobacteria depends on proteolysis of two essential enzymes, namely, LpxC and KdtA, by the membrane-bound AAA (ATPases associated with various cellular activities) protease FtsH (8,9).…”

mentioning

confidence: 99%

“…LpxC is a deacetylase, which catalyzes the committed step in biosynthesis of lipid A (10) and KdtA attaches two 3-deoxy-D-manno-octulosonate (Kdo) residues to lipid IV A to form Kdo 2 -lipid A, the minimal structure of LPS that is essential for survival (11). The C terminus of LpxC has been shown to contain a nonpolar, sequence-and length-specific signal for degradation by the FtsH protease (6,7). Besides its essential role in controlling LPS biosynthesis, FtsH is involved in the quality control of membrane proteins and the regulation of various cellular mechanisms, in particular under stress conditions (12)(13)(14)(15).…”

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confidence: 99%

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FtsH-Mediated Coordination of Lipopolysaccharide Biosynthesis in Escherichia coli Correlates with the Growth Rate and the Alarmone (p)ppGpp

2013

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The outer membrane is the first line of defense for Gram-negative bacteria and serves as a major barrier for antibiotics and other harmful substances. The biosynthesis of lipopolysaccharides (LPS), the essential component of the outer membrane, must be tightly controlled as both too much and too little LPS are toxic. In Escherichia coli, the cellular level of the key enzyme LpxC, which catalyzes the first committed step in LPS biosynthesis, is adjusted by proteolysis carried out by the essential and membrane-bound protease FtsH. Here, we demonstrate that LpxC is degraded in a growth rate-dependent manner with half-lives between 4 min and >2 h. According to the cellular demand for LPS biosynthesis, LpxC is degraded during slow growth but stabilized when cells grow rapidly. Disturbing the balance between LPS and phospholipid biosynthesis in favor of phospholipid production in an E. coli strain encoding a hyperactive FabZ protein abolishes growth rate dependency of LpxC proteolysis. Lack of the alternative sigma factor RpoS or inorganic polyphosphates, which are known to mediate growth rate-dependent gene regulation in E. coli, did not affect proteolysis of LpxC. In contrast, absence of RelA and SpoT, which synthesize the alarmone (p)ppGpp, deregulated LpxC degradation resulting in rapid proteolysis in fast-growing cells and stabilization during slow growth. Our data provide new insights into the essential control of LPS biosynthesis in E. coli.

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Section: Methodsmentioning

confidence: 99%

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confidence: 99%

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FtsH-Mediated Coordination of Lipopolysaccharide Biosynthesis in Escherichia coli Correlates with the Growth Rate and the Alarmone (p)ppGpp

2013

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“…LpxC catalyzes the rate-limiting step in lipid A biosynthesis (3). In E. coli, this step was shown to be regulated by FtsH-mediated proteolysis of LpxC, which requires a specific signal (LAXXXXXAVLA) at the C terminus of LpxC (38)(39)(40). This signal is not present at the C terminus of meningococcal LpxC.…”

Section: Discussionmentioning

confidence: 99%

“…4A), consistent with their translucent-colony phenotype. In E. coli, LpxC catalyzes the rate-limiting step in LPS synthesis, and its levels are carefully controlled at the posttranscriptional level (38)(39)(40). Hence, we considered the possibility that Ght regulates LpxC activity in N. meningitidis and that its absence can be compensated for by lpxC overexpression.…”

Section: Phenotype Of a Ght Mutantmentioning

confidence: 99%

Ght Protein of Neisseria meningitidis Is Involved in the Regulation of Lipopolysaccharide Biosynthesis

et al. 2014

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Lipopolysaccharide (LPS) is a major component of the outer membrane of Gram-negative bacteria and is responsible for the barrier function of this membrane. A ght mutant of Neisseria meningitidis that showed increased sensitivity to hydrophobic toxic compounds, suggesting a breach in this permeability barrier, was previously described. Here, we assessed whether this phenotype was possibly caused by a defect in LPS transport or synthesis. The total amount of LPS appeared to be drastically reduced in a ght mutant, but the residual LPS was still detected at the cell surface, suggesting that LPS transport was not impaired. The ght mutant was rapidly overgrown by pseudorevertants that produced normal levels of LPS. Genetic analysis of these pseudorevertants revealed that the lpxC gene, which encodes a key enzyme in LPS synthesis, was fused to the promoter of the upstreamlocated pilE gene, resulting in severe lpxC overexpression. Analysis of phoA and lacZ gene fusions indicated that Ght is an inner membrane protein with an N-terminal membrane anchor and its bulk located in the cytoplasm, where it could potentially interact with LpxC. Cell fractionation experiments indeed indicated that Ght tethers LpxC to the membrane. We suggest that Ght regulates LPS biosynthesis by affecting the activity of LpxC. Possibly, this mechanism acts in the previously observed feedback inhibition of LPS synthesis that occurs when LPS transport is hampered.

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In vivo trapping of FtsH substrates by label‐free quantitative proteomics

et al. 2016

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FtsH is the only membrane-bound and essential protease in Escherichia coli. It is responsible for the degradation of regulatory proteins and enzymes such as the heat-shock sigma factor RpoH or LpxC, the key enzyme of lipopolysaccharide biosynthesis. To find new FtsH targets, we trapped substrates in E. coli cells from exponential and stationary growth phase by using a proteolytically inactive FtsH variant. Subsequent analysis of the isolated FtsH-substrate complexes by label-free nanoLC-MS/MS revealed more than 50 putative FtsH substrates, among them five already known substrates. Four out of thirty-seven tested candidates were found to be novel FtsH substrates as shown by in vivo degradation experiments. Six other candidates were degraded by one or more other protease(s). The FtsH substrates SecD and ExbD are involved in transport processes across the membrane, whereas the physiological roles of YlaC and YhbT are yet unknown. The presence of the previously identified YfgM degron in two of the novel substrates suggests general rules for substrate recognition of this unique protease.

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Sequence and Length Recognition of the C-terminal Turnover Element of LpxC, a Soluble Substrate of the Membrane-bound FtsH Protease

Cited by 43 publications

References 52 publications

FtsH-Mediated Coordination of Lipopolysaccharide Biosynthesis in Escherichia coli Correlates with the Growth Rate and the Alarmone (p)ppGpp

FtsH-Mediated Coordination of Lipopolysaccharide Biosynthesis in Escherichia coli Correlates with the Growth Rate and the Alarmone (p)ppGpp

Ght Protein of Neisseria meningitidis Is Involved in the Regulation of Lipopolysaccharide Biosynthesis

In vivo trapping of FtsH substrates by label‐free quantitative proteomics

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