Abstract
YidC plays a role in the integration and assembly of many (if not all) Escherichia coli inner membrane proteins. Strikingly, YidC operates in two distinct pathways: one associated with the Sec translocon that also mediates protein translocation across the inner membrane and one independent from the Sec translocon. YidC is homologous to Alb3 and Oxa1 that function in the integration of proteins into the thylakoid membrane of chloroplasts and inner membrane of mitochondria, respectively. Here, we have expressed the conserved region of yeast Oxa1 in a conditional E. coli yidC mutant. We find that Oxa1 restores growth upon depletion of YidC. Data obtained from in vivo protease protection assays and in vitro cross-linking and folding assays suggest that Oxa1 complements the insertion of Sec-independent proteins but is unable to take over the Sec-associated function of YidC. Together, our data indicate that the Sec-independent function of YidC is conserved and essential for cell growth.
Highlights
YidC plays a role in the integration and assembly of many Escherichia coli inner membrane proteins
EcOxa1 Is Expressed in E. coli and Complements the Growth Defect of a Conditional yidC Strain—Members of the Alb3/ Oxa1/YidC family are ubiquitous membrane proteins that share a similar topology with a conserved core of five transmembrane segments (TMs) separated by relatively short loops (Fig. 1A)
E. coli YidC is a member of the conserved Alb3/Oxa1/YidC protein family found throughout prokaryotes and eukaryotes [1,2,3,4]
Summary
Plasmid pEH1-ecOxa1His harbors the ecOxa fusion construct with a N-terminal hexahistidinyl tag under control of a lac promoter For construction of this plasmid, the sequence encoding residues 2–247 of YidC was amplified by PCR from DNA isolated from E. coli DH5␣. For F0c protease accessibility assays, the temperature sensitive YidC depletion strain KO1672 and its isogenic control strain KO1670 harboring pCL-ecOxa together with pBAD24-F0cHA were grown as described [29]. Both strains were grown overnight at 30 °C in LB medium. To deplete KO1672 for YidC, cells were grown at 42 °C, and for the expression of the ecOxa construct IPTG was added to the medium as indicated. Samples were analyzed as described [33]. mAb4B1 and mAb4B11 were purified as described [19]
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