Membrane proteins present more than 25% of the proteome of all cells, and mediate the cell's interaction with its environment. They are of prime pharmaceutical interest as they constitute ~50% of known and potential new drug targets. Understanding the function and molecular structure of this class of proteins is of key interest. A large proportion of proteins needs to be incorporated into specific cellular membranes; in <i>E. coli</i>, it is about 20-30% of the total number. This process is known as protein translocation. Several membrane proteins are involved in protein quality control mechanisms during this process. FtsH is a conserved bacterial protease that degrades improperly folded integral membrane proteins using an ATP-dependent mechanism. An integral membrane protein, YccA, comprising seven transmembrane regions, is one of the substrates of FtsH and inhibits the FtsH proteolytic activity. SecY is degraded by FtsH when it is not in a complex with SecE and SecG. The membrane protein YccA, inhibits FtsH, and counteracts this destruction, when it is overproduced or stabilized. In the mutant form of YccA lacking eight amino acids in the N-terminal end, SecY was found to be stable. This mutant protein, YccA11, binds to FtsH, but cannot be degraded by FtsH, unlike wild-type YccA. In this study, we cloned both YccA and YccA11 from the <i>E. coli </i>K-12 strain genomic DNA using Gibson assembly method with a His-tag on the N-terminal end, and overexpressed in E. coli. Membranes were solubilized with either DDM or LMNG. Proteins were purified by affinity chromatography. The size and purity of YccA was confirmed by SDS-PAGE, and size exclusion chromatography. The stability of YccA was investigated by cross-linking with BS3 (bis(sulfosuccinimidyl)suberate), also its interaction with FtsH was investigated using cross-linking mass spectrometry with DSBU (Disuccinimidyl Dibutyric Urea). Furthermore, the degradation of YccA was studied by the recombinant FtsH. The outcomes will help structural studies and understanding of the mechanism behind this fundamental process.

Insights into bacterial membrane protein quality control: YccA and its interaction with FtsH

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ACS Fall 2022

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