The interaction network of the YidC insertase with the SecYEG translocon, SRP and the SRP receptor FtsY

Abstract

YidC/Oxa1/Alb3 are essential proteins that operate independently or cooperatively with the Sec machinery during membrane protein insertion in bacteria, archaea and eukaryotic organelles. Although the interaction between the bacterial SecYEG translocon and YidC has been observed in multiple studies, it is still unknown which domains of YidC are in contact with the SecYEG translocon. By in vivo and in vitro site-directed and para-formaldehyde cross-linking we identified the auxiliary transmembrane domain 1 of E. coli YidC as a major contact site for SecY and SecG. Additional SecY contacts were observed for the tightly packed globular domain and the C1 loop of YidC, which reveals that the hydrophilic cavity of YidC faces the lateral gate of SecY. Surprisingly, YidC-SecYEG contacts were only observed when YidC and SecYEG were present at about stoichiometric concentrations, suggesting that the YidC-SecYEG contact in vivo is either very transient or only observed for a very small SecYEG sub-population. This is different for the YidC-SRP and YidC-FtsY interaction, which involves the C1 loop of YidC and is efficiently observed even at sub-stoichiometric concentrations of SRP/FtsY. In summary, our data provide a first detailed view on how YidC interacts with the SecYEG translocon and the SRP-targeting machinery.

Protein Interactomes Identify Distinct Pathways forStreptococcus mutansYidC1 and YidC2 Membrane Protein Insertases

“…However, that study utilized deletion mutants while the current study is more indicative of protein interactions in the wild-type strain. The identification of SecY and YajC, as well as YidC1, in the lower region suggests that these represent components of a pathway that operates independently of YidC2 and/or the SRP, and supports previous reports of a SecY-YidC interaction in E. coli that is modulated by YajC [15, 17, 36, 41]. Thus in S. mutans , YidC1 likely serves as the major interaction partner of SecY/YajC.…”

“…Thus in S. mutans , YidC1 likely serves as the major interaction partner of SecY/YajC. It has also been reported in E. coli that SecYEG and YidC compete for binding to the SRP receptor, FtsY [17]. Our results suggest, therefore, that YidC1 and YidC2 participate in two distinct pathways such that YidC1 functions in concert with the SecY translocon independently of the SRP pathway, while YidC2 functions primarily in concert with the SRP pathway.…”

“…That is, the immunocapture dataset was likely biased against proteins that react with cytoplasmic loop 1 or the YidC2 C-terminal tail. In E. coli , cytoplasmic loop 1 of YidC has been reported to interact with SecY in an in vivo photo-crosslinking assay [17]. Thus, occupancy of the corresponding loop in YidC2 by SecY could potentially have blocked reactivity with the anti-YidC2 loop antibody and explain why YajC, but not SecY, was identified in the immunocapture assay.…”

“…E.coli YidC can work independently [11, 13, 14], in collaboration with the Sec machinery [15-17], and in collaboration with SRP pathway components [17, 18]. E.coli membrane proteins inserted by YidC alone are relatively few, and generally contain only one or two TM domains [19-24].…”

“…Past studies of Gram-positive YidCs have used genetic approaches comparing phenotypic differences between wild-type and mutant strains [7, 8, 12], or cross-complementation in heterologous systems [26-28]. Solved crystal structures of bacterial YidCs [29-35] have also facilitated investigations of insertase interactions with other protein transport machinery components [17, 36-40]. Such studies employed in vitro or highly defined systems and provided information regarding particular protein-protein combinations.…”