Structural basis of Sec-independent membrane protein insertion by YidC

Abstract

Newly synthesized membrane proteins must be accurately inserted into the membrane, folded and assembled for proper functioning. The protein YidC inserts its substrates into the membrane, thereby facilitating membrane protein assembly in bacteria; the homologous proteins Oxa1 and Alb3 have the same function in mitochondria and chloroplasts, respectively. In the bacterial cytoplasmic membrane, YidC functions as an independent insertase and a membrane chaperone in cooperation with the translocon SecYEG. Here we present the crystal structure of YidC from Bacillus halodurans, at 2.4 Å resolution. The structure reveals a novel fold, in which five conserved transmembrane helices form a positively charged hydrophilic groove that is open towards both the lipid bilayer and the cytoplasm but closed on the extracellular side. Structure-based in vivo analyses reveal that a conserved arginine residue in the groove is important for the insertion of membrane proteins by YidC. We propose an insertion mechanism for single-spanning membrane proteins, in which the hydrophilic environment generated by the groove recruits the extracellular regions of substrates into the low-dielectric environment of the membrane.

Hydrophilic microenvironment required for the channel-independent insertase function of YidC protein

“…The other B. subtilis YidC homolog, YidC2 (YqjG), also functions by means of a similar mechanism for insertion of MifM (12). The importance of the cavity was also supported by photo-cross-linking experiments showing that the inner surface of the cavity of SpoIIIJ interacts with substrate protein in vivo (21). Together with the crystal structure of E. coli YidC (22), it is suggested that having a hydrophilic and positively charged cavity is a feature shared by the YidC family members.…”

“…In our previous genetic studies, single Ala substitutions for the conserved hydrophilic residues in the cavity did not deteriorate SpoIIIJ functions, except for Arg73 (21). We reason that single Ala substitutions may be insufficient to reduce the overall hydrophilicity of the cavity.…”

“…Although the SecYEG translocon overcomes this difficulty by forming a polypeptide-conducting channel that sequesters a translocating polypeptide from the lipidic environment (15)(16)(17), several lines of evidence (19,20,27), most notably the crystal structure of B. halodurans YidC2 (21), suggest that YidC uses a channel-independent mechanism.…”

“…The SpoIIIJ cavity contains an Arg residue that is functionally essential, leading us to propose a charge attraction model for the initiation of translocation of MifM-like substrates (21). In this case, substrate recognition may not be based on strict structural complementarity and electrostatic interaction may allow certain positional flexibility.…”

“…negatively charged residues in the extracytoplasmic and TM regions of MifM are essential for insertion of MifM into the membrane (21). From these results, we proposed that SpoIIIJ mediates insertion of a class of membrane proteins such as MifM by a channel-independent mechanism, in which electrostatic attraction between the SpoIIIJ cavity and the substrate initiates the reaction (21). The other B. subtilis YidC homolog, YidC2 (YqjG), also functions by means of a similar mechanism for insertion of MifM (12).…”