In bacteria, it has been widely recognized that SecYEG translocase is the main protein-conducting channel that mediates the insertion of the inner membrane proteins and also the membrane translocation of the exported proteins to the periplasmic space. Recently a new inner membrane protein, YidC, was discovered to be involved in membrane protein insertion. YidC works in conjunction with the Sec translocase and function in integrating proteins into the lipid bilayer, or as a chaperone to fold the proteins into their correct functional structure. YidC can also function on its own as an insertase to insert inner membrane proteins independent of the Sec translocase. In my research, I focused on the membrane insertion of proteins in E.coli using these two pathways.
In chapter 2, we describe two new yidC mutants that lead to a cold-sensitive phenotype in bacterial cell growth. Both alleles impart a cold sensitive phenotype that result from point mutations localized to the third transmembrane segment of YidC, indicating this region is crucial for YidC function. We found that the yidCC423R mutant confers a weak phenotype on membrane protein insertion while a yidCP431L mutant leads to a stronger phenotype. In both cases, the affected substrates include the Pf3 coat protein and the F0C (subunit c of the F 1F0 ATP synthase); CyoA (the quinol binding subunit of the cytochrome bo3 quinol oxidase complex) and wild type procoat are slightly or not affected in either cold-sensitive mutant. To determine if the different substrates require varying levels of YidC activity for membrane insertion, we performed studies where YidC was depleted using an arabinose-dependent expression system. We found that -3M-PC-Lep and Pf3 P2 required the highest amount of YidC, and CyoA-N-P2 and PC-Lep required the least while FoC required moderate YidC levels. Although the cold-sensitive mutations can preferentially affect one substrate over another, our results indicate that different substrates require different levels of YidC activity for membrane insertion. Finally, we obtained several intragenic suppressors that overcome the cold-sensitivity of the C423R mutation. One pair of mutations suggests an interaction between TM2 and TM3 of YidC. The studies reveal the critical regions of the YidC protein and provide insight into the substrate profile of the YidC insertase.
In chapter 3, we proposed a new hypothesis explaining why some M13 Procoat mutants are Sec translocase dependent. Such M13 procoat mutants are proposed to bind to SecA in vivo after synthesis and are targeted to the Sec translocase for membrane insertion. In this case, membrane insertion of the M13 procoat mutant is dependent on Sec pathway. However, when SecA is not present, the M13 procoat mutant will be targeted to YidC and use the YidC only pathway for the membrane insertion. We utilized a SecA depletion strain to test our hypothesis in vivo. Different Sec dependent PCLep mutants and Sec independent PCLep mutants were studied under the condition of SecA depletion or SecA inhibition. We show that the Sec dependent proteins -5PCLep, -3MPCLep and 3NPCLep can be inserted into the inner membrane by YidC only pathway when the SecA was depleted.
|Commitee:||Cowan, James, Dean, Donald, Jhiang, Sissy|
|School:||The Ohio State University|
|School Location:||United States -- Ohio|
|Source:||DAI-B 78/11(E), Dissertation Abstracts International|
|Keywords:||Cold sensitive, Membrane protein, Sec pathway, Seca, Yidc|
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