Interaction of Streptococcus mutans YidC1 and YidC2 with Translating and Nontranslating Ribosomes

Abstract

The YidC/OxaI/Alb3 family of membrane proteins is involved in the biogenesis of integral membrane proteins in bacteria, mitochondria, and chloroplasts. Gram-positive bacteria often contain multiple YidC paralogs that can be subdivided into two major classes, namely, YidC1 and YidC2. The Streptococcus mutans YidC1 and YidC2 proteins possess C-terminal tails that differ in charges (؉9 and ؉ 14) and lengths (33 and 61 amino acids). The longer YidC2 C terminus bears a resemblance to the C-terminal ribosome-binding domain of the mitochondrial OxaI protein and, in contrast to the shorter YidC1 C terminus, can mediate the interaction with mitochondrial ribosomes. These observations have led to the suggestion that YidC1 and YidC2 differ in their abilities to interact with ribosomes. However, the interaction with bacterial translating ribosomes has never been addressed. Here we demonstrate that Escherichia coli ribosomes are able to interact with both YidC1 and YidC2. The interaction is stimulated by the presence of a nascent membrane protein substrate and abolished upon deletion of the C-terminal tail, which also abrogates the YidC-dependent membrane insertion of subunit c of the F 1 F 0 -ATPase into the membrane. It is concluded that both YidC1 and YidC2 interact with ribosomes, suggesting that the modes of membrane insertion by these membrane insertases are similar.

A YidC-like Protein in the Archaeal Plasma Membrane

“…Total 70S E. coli ribosomes were purified according to a published protocol (Wu et al, 2013) with the exception that we used E. coli BL21(DE3) cells. Stalled RNCs were obtained using a construct containing a 3× Strep-tag II sequence fused to the first transmembrane segment of the YidC substrate F 0 c and to the SecM stalling sequence, similar to a previously utilized construct (Kedrov et al, 2013).…”

“…An artificially synthesized gene was Gibson assembled into pET21a and transformed into BL21(DE3) cells. Stalled RNC-F 0 cs were expressed and purified according to the published protocol (Wu et al, 2013) (Figure S4). Both the total 70S ribosomes and the stalled RNCs were quantified by A 260 , flash frozen, and stored at −80°C.…”

Structural Dynamics of the YidC:Ribosome Complex during Membrane Protein Biogenesis

“…A monomer of YidC interacts with translating ribosomes both in the detergent environment and in the lipid bilayer (Kedrov et al., 2013, Wickles et al., 2014), thus representing the functional insertase unit. Similar to the SecYEG system, YidC specifically interacts with ribosomes that expose hydrophobic nascent chains (Kedrov et al., 2013, Wu et al., 2013). The positively charged C terminus and a short cytoplasmic loop connecting TM4 and TM5 facilitate this interaction (Geng et al., 2015), while the YidC variant lacking the C terminus (YidCΔC) is impaired in ribosome binding (Kedrov et al., 2013).…”

Role of the Cytosolic Loop C2 and the C Terminus of YidC in Ribosome Binding and Insertion Activity

“…The C-terminal regions of the mitochondrial Oxa1 and Streptococcus mutants YidC1 and YidC2 have been shown to be crucial for the contact with ribosomes (21)(22)(23), and deletions within these domains compromised the protein insertion function. In E. coli, the C terminus of YidC is positively charged, but it is substantially shorter (13 residues; total charge ϩ5) in comparison with Oxa1 (86 residues; ϩ14), YidC1 (33 residues; ϩ9), and YidC2 (61 residues; ϩ14) (19,22,23). Recently, we have investigated the interaction of YidC with translation-stalled ribosomes by means of the fluorescence correlation spectroscopy (FCS) technique under physiological condition using nanodisc-embedded YidC to mimic the native lipids environment (24).…”