Studies on Escherichia coli YidC insertase during membrane protein insertion

In bacteria, it has been widely recognized that SecYEG translocon is the major translocase used to insert proteins into or export proteins across the membrane bilayer. It has also been found that SecYEG translocase helps proteins adopt their membrane topology and proper conformation. Recent studies have added another pathway into the paradigm. The Oxa1/Alb3/YidC family is a group of evolutionarily conserved proteins existing in mitochondria, chloroplasts, and bacteria. The Oxa1/Alb3/YidC pathway is believed to independently insert proteins into membrane bilayer and facilitate their subunits assembly in the membrane. In addition, the Oxa1/Alb3/YidC proteins can assist the Sec pathway by helping release the membrane proteins from the Sec translocase channel and moving them laterally into the lipid bilayer. In this research, there are two objectives. First, the interaction of YidC with the SecDF components of the SecYEG translocase was studied in details. The specific region responsible for the interaction was evaluated and the importance of YidC-SecDF binding in membrane protein insertion was investigated. Second, the features of the YidC substrates that promote membrane protein insertion were studied. In particular, the hydrophobicity threshold converting a YidC substrate to Sec pathway and the individual contribution of the twenty amino acids for transmembrane insertion was measured.; In chapter 2, we investigated the interaction of YidC with SecDF of the SecYEG translocase. YidC was previously discovered to interact with Sec translocase using SecDF as a bridge. SecD and SecF are accessory proteins in the SecYEG complex that facilitate membrane insertion but their exact function is not known. To address the importance of the YidC-SecDF interaction, we constructed a series of deletion mutants and tested their binding efficiency. The co-purification experiments showed that the loss of amino acids 24 to 264 causes a defect in YidC binding to SecDF. Then we cloned different sequences within this region of YidC and tested the ability of those sequences to bind to SecDF in vitro. In this study we found that the amino acid segment 215 to 265 is able to interact with SecDF. In addition, we investigated the membrane insertion of some "YidC only" substrates and YidC/SecYEG substrates under these deletion mutants. To our surprise, the YidC mutant lacking residues 24 to 264 can insert both substrates into the membrane very well. This result suggests that the interacting with SecDF is not critical to the function of YidC in the SecYEG pathway. Another possibility is that the high amount of YidC compensates the low binding affinity. The natural copies of YidC and SecYEG in E. coli cells are at a ratio of about 250 to one.; Presently, only a few membrane proteins have been confirmed to be a YidC pathway substrates. The transmembrane domains of YidC substrates were shown to physically contact the YidC molecules during the membrane insertion process by crosslinking studies. We assumed that hydrophobic interaction is the force bringing the two molecules together. To quantify the hydrophobic force we set up a model system. Two endonuclease sites were engineered at both sides of the gene encoding transmembrane domain of M13 procoat, so we can replace the transmembrane domain with the artificial sequences. The theoretical apparent free energy of twenty amino acids was measured using this system. A 19 amino acid segment consisting of only alanine, which is not considered a hydrophobic amino acid, is able to insert into and anchor the protein into the membrane bilayer. The 19 amino acids stretch of tyrosine, which is more hydrophilic as shown in our hydrophobicity scale, is unable to be inserted into membrane. When the artificial segment becomes even more hydrophilic, as the 19 serine stretch, this protein domain is secreted across membrane via SecYEG pathway. We also determined that the shortest hydrophobic domain to be inserted into membrane is a...