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Different pathways for protein degradation by the FtsH/HflKC membrane-embedded protease complex: an implication from
1Department of Cell Biology, Institute for Virus Research, Kyoto University, Japan.
Abstract:
Escherichia coli FtsH (HflB) is a membrane-bound and ATP-dependent zinc-metalloproteinase, which forms a complex with a pair of periplasmically exposed membrane proteins, HflK and HflC. It is the protease that degrades uncomplexed forms of the SecY subunit of protein translocase. Here, we characterized a new class of SecY-stabilizing mutation on the E. coli chromosome. The mutation (yccA11) is an internal deletion within a gene (yccA) known as an open reading frame for a hydrophobic protein with putative seven transmembrane segments. The YccA protein was found to be degraded in an FtsH-dependent manner in vivo and in vitro, whereas the YccA11 mutant protein, lacking eight amino acid residues within the amino-terminal cytoplasmic domain, was refractory to the degradation. The yccA11 mutation exhibited partial dominance when overexpressed. Cross-linking, co-immunoprecipitation, and histidine tagging experiments showed that YccA11 as well as YccA can associate with both the FtsH and the HflKC proteins. Thus, the mutant YccA protein appeared to compete with SecY for recognition by the FtsH proteolytic system and the residues deleted by the yccA mutation are required for the initiation of proteolysis by FtsH. Interestingly, the inhibitory action of YccA11 was mediated by HflKC, since the deletion of hflK-hflC suppressed the yccA11 phenotype. The yccA11 mutation stabilized subunit a of the proton ATPase F0 segment as well, but not the CII protein of bacteriophage lambda or the sigma 32 protein. From these results we suggest that there are at least two pathways for FtsH-dependent protein degradation, only one of which (probably for membrane proteins) is subject to the HflKC-dependent interference by the YccA11 mutant substrate.
Insights
A mutation in the E. coli yccA gene creates a mutant protein that stabilizes SecY by interfering with the FtsH protease system. This interference is mediated by HflKC proteins, suggesting distinct FtsH degradation pathways.
Area of Science:
- Molecular biology
- Protein degradation
- Membrane protein regulation
Background:
- Escherichia coli FtsH is an ATP-dependent metalloproteinase that degrades uncomplexed SecY, a subunit of the protein translocase.
- FtsH functions in a complex with membrane proteins HflK and HflC.
Purpose of the Study:
- To characterize a novel mutation in E. coli that stabilizes SecY.
- To investigate the mechanism by which the mutant protein interacts with the FtsH proteolytic system.
Main Methods:
- Genetic analysis of a yccA deletion mutation (yccA11).
- In vivo and in vitro degradation assays.
- Protein interaction studies including cross-linking and co-immunoprecipitation.
- Analysis of suppressor mutations in hflK-hflC.
Main Results:
- The yccA11 mutation, an internal deletion in a hydrophobic protein gene, confers SecY stability.
- The mutant YccA11 protein is resistant to FtsH-dependent degradation and competes with SecY for FtsH binding.
- The inhibitory effect of YccA11 on FtsH is dependent on the HflKC complex.
- YccA11 stabilizes other proteins like subunit a of proton ATPase F0, but not bacteriophage lambda CII or sigma 32.
Conclusions:
- The yccA11 mutation reveals a mechanism for modulating FtsH activity through substrate competition.
- The HflKC complex plays a crucial role in mediating the interaction between YccA11 and FtsH.
- These findings suggest the existence of at least two distinct FtsH-dependent protein degradation pathways.