The Conserved Third Transmembrane Segment of YidC Contacts Nascent Escherichia coli Inner Membrane Proteins

Abstract

Escherichia coli YidC is a polytopic inner membrane protein that plays an essential and versatile role in the biogenesis of inner membrane proteins. YidC functions in Sec-dependent membrane insertion but acts also independently as a separate insertase for certain small membrane proteins. We have used a site-specific cross-linking approach to show that the conserved third transmembrane segment of YidC contacts the transmembrane domains of both nascent Sec-dependent and -independent substrates, indicating a generic recognition of insertion intermediates by YidC. Our data suggest that specific residues of the third YidC transmembrane segment ␣-helix is oriented toward the transmembrane domains of nascent inner membrane proteins that, in contrast, appear quite flexibly positioned at this stage in biogenesis.

Conserved Negative Charges in the Transmembrane Segments of Subunit K of the NADH:Ubiquinone Oxidoreductase Determine Its Dependence on YidC for Membrane Insertion

“…YidC has been shown to contact the TMSs of numerous integral membrane proteins in various cross-linking studies (24,42,43), and yet YidC is dispensable in the insertion of most of these proteins (26,44,45). Using in vitro insertion into proteoliposomes, we show that NuoK minimally requires both YidC and SecYEG for insertion.…”

“…CyoA, however, does not contain any membrane-negative charges. YidC is not essential for the insertion of FtsQ (26) and LepB (49), even though interaction between these proteins and YidC during membrane insertion has been observed (1,24,42,43,50). FtsQ does not contain any membrane-embedded charges, whereas LepB contains a glutamate in TM2.…”

The YidC/Oxa1/Alb3 protein family: common principles and distinct features

“…However, further mutational analysis revealed a large promiscuity of the core residues . The importance of core TMS2 (TMS3 in E. coli YidC) is also evident from chemical cross-linking to known YidC substrates (Klenner et al , 2008 ;Yu et al , 2008 ) all across the membrane helix, N-terminal domain is composed of an additional TMS followed by a large periplasmatic loop. Recently, the crystal structure of the periplasmatic domain of E. coli YidC was solved (Oliver and Paetzel , 2008 ;Ravaud et al , 2008 ).…”

Role for Escherichia coli YidD in Membrane Protein Insertion

“…Truncated mRNA was prepared from HindIII-linearized plasmid pC4Meth108FtsQCys15, -36, and -61, which introduces a Cys residue in FtsQ at positions 15, 36, and 61, respectively, as described previously (36). In vitro translation, insertion in IMVs, cross-linking with BMOE, and sodium carbonate extraction were carried out as described previously (55). Immunoprecipitations (IPs) on sodium carbonate-extracted samples were carried out as described previously (45).…”

“…FtsQ is a type II IMP that spans the IM once and plays a critical role in cell division. The TM of the well-characterized 108FtsQ intermediate is located between residues 24 and 49, exposed outside the ribosome, and has been shown to interact with YidC (36,55). Because YidD contains three conserved cysteine residues, we decided to use sulfhydryl-specific cross-linking upon introduction of single cysteine residues in 108FtsQ to probe a putative interaction.…”