Identification of YidC Residues That Define Interactions with the Sec Apparatus

Abstract

In bacteria, a subset of membrane proteins insert into the membrane via the Sec apparatus with the assistance of the widely conserved essential membrane protein insertase YidC. After threading into the SecYEG translocon, transmembrane segments of nascent proteins are thought to exit the translocon via a lateral gate in SecY, where YidC facilitates their transfer into the lipid bilayer. Interactions between YidC and components of the Sec apparatus are critical to its function. The first periplasmic loop of YidC interacts directly with SecF. We sought to identify the regions or residues of YidC that interact with SecY or with additional components of the Sec apparatus other than SecDF. Using a synthetic lethal screen, we identified residues of YidC that, when mutated, led to dependence on SecDF for viability. Each residue identified is highly conserved among YidC homologs; most lie within transmembrane domains. Overexpression of SecY in the presence of two YidC mutants partially rescued viability in the absence of SecDF, suggesting that the corresponding wild-type YidC residues (G355 and M471) participate in interactions, direct or indirect, with SecY. Staphylococcus aureus YidC complemented depletion of YidC, but not of SecDF, in Escherichia coli. G355 of E. coli YidC is invariant in S. aureus YidC, suggesting that this highly conserved glycine serves a conserved function in interactions with SecY. This study demonstrates that transmembrane residues are critical in YidC interactions with the Sec apparatus and provides guidance on YidC residues of interest for future structure-function analyses.

A central cavity within the holo-translocon suggests a mechanism for membrane protein insertion

“…3c). The resulting proximity of the YidC TMD to SecDF is supported by recent independent screening and complementation experiments31. The YidC TMD was suggested to facilitate membrane-protein insertion, borne out by cross-linking between nascent TM-helices successively to SecY, lipids and finally to YidC10, including YidC-TM332.…”

“…Here, we combined a range of biophysical data to provide mechanistic and structural insights into the HTL membrane protein complex in an integrative approach. Based on the SANS data, EM volumes, subunit localization experiments and available biochemical data345122731 we present a quasi-atomic model of HTL and a working model for HTL function in translocation into and across the membrane and membrane protein folding (Fig. 4).…”

Alternative Evolutionary Pathways for Drug-Resistant Small Colony Variant Mutants in Staphylococcus aureus

“…The sequence analysis (see Table S1 in the supplemental material) showed that each of the menadione-auxotrophic SCVs carried a mutation in one of the menadione biosynthesis genes ( menA , menB , or menE ) and that each of the hemin-auxotrophic SCVs carried a mutation in one of the hemin biosynthesis genes ( hemB , hemC , hemD , hemE , or hemH ). The single nonauxotrophic SCV carried mutations in qoxB , oxaA , srrA , and ftsQ , genes that are involved in respiration or cell division ( 26 – 31 ), possibly explaining the SCV phenotype of this mutant. The median kanamycin MIC of the 26 mutant strains is 48 mg/liter ( Table S1 ).…”

YidC/Alb3/Oxa1 Family of Insertases

“…Additional residues in this or other regions may also be important for the YidC and SecY interaction. Recently, Li et al (75) discovered Gly-355 in TM2 and Met-471 in TM4 of the E. coli YidC to be important for YidC-SecY interaction using a synthetic lethal screen. It has been hypothesized that the interaction of YidC with SecYEG is facilitated by SecDFYajC (76).…”