Consecutive Steps of Membrane Insertion of the Two-spanning MscL Protein by Insertase YidC

Abstract

A fundamental problem in biology is understanding how membrane proteins are inserted and assembled into their three-dimensional structures. The YidC/Oxa1/Alb3 insertases, found in bacteria, mitochondria, and chloroplasts play crucial roles in membrane protein insertion. In this study, we investigated the YidC-mediated insertion of MscL, a 2-spanning membrane protein by analyzing a series of translational arrested intermediates and probing the interactions with YidC using thio-crosslinking. Our findings reveal that the first TM segment and the second TM segment of MscL interact cotranslationally with the YidC membrane-embedded greasy slide, although in a delayed manner. The translocation of the periplasmic loop in between the two TM segments only occurs after TM2 engages with the greasy slide of YidC, showing that full insertion occurs late during synthesis. Remarkably, TM2 does not displace TM1 from the slide, and the contact is maintained even when the full-length protein emerges from the ribosome. These results demonstrate a well-ordered sequence of events during the membrane insertion of multi-spanning membrane proteins, providing new insights into the mechanistic role of YidC in protein assembly.