Direct Interaction of YidC with the Sec-independent Pf3 Coat Protein during Its Membrane Protein Insertion

Abstract

YidC is a newly defined translocase component that mediates the insertion of proteins into the membrane bilayer. How YidC functions in the insertion process is not known. In this study, we report that the Sec-independent Pf3 coat protein requires the YidC protein specifically for the membrane translocation step. Using photocrosslinking techniques and ribosome-bound Pf3 coat derivatives with an extended carboxyl-terminal region, we found that the transmembrane region of the Pf3 coat protein physically interacts with YidC and the bacterial signal recognition particle Ffh component. We also find that in the insertion pathway, Pf3 coat interacts strongly with YidC only after its transmembrane segment is fully exposed outside the ribosome tunnel. Interaction between Pf3 coat and YidC occurs even in the absence of the proton motive force and with a Pf3 coat mutant that is defective in transmembrane insertion. Our study demonstrates that YidC can directly interact with a Sec-independent membrane protein, and the role of YidC is at the stage of folding the Pf3 protein into a transmembrane configuration.

Defining the Regions of Escherichia coli YidC That Contribute to Activity

“…YidC has been found associated with the SecYEGDF-YajC complex and to interact with Sec-dependent membrane proteins during membrane insertion (3)(4)(5). However, Escherichia coli depleted of YidC is mildly defective in the insertion of Sec-dependent membrane proteins (6,7), but is blocked in the insertion of proteins that do not use the Sec translocase, such as the M13 phage procoat protein and the Pf3 phage coat protein (6,8,9). These Sec-independent proteins were previously thought not to require a proteinaceous factor (10 -12).…”

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

“…Escherichia coli YidC is an abundant membrane protein, with ϳ2500 copies per cell (1) and it is involved in the insertion, folding and/or assembly of membrane proteins into the cytoplasmic membrane independently or in concert with the SecYEG translocon (4). E. coli YidC is essential for cell viability (5) and has been shown to function as an insertase in the membrane insertion of the filamentous phage Pf3 coat and M13 pro-coat proteins (5,6) and the endogenous substrates F 0 c (7), MscL (8,9), and TssL (10). In cooperation with the Sec translocase, YidC assists in the membrane insertion of CyoA (11,12), NuoK (13), and F 0 a and F 0 b subunits of F 1 F 0 ATPase (14), and the translocation of the periplasmic loop 1 and loop 2 of TatC (15).…”

Novel GFP Expression Using a Short N-Terminal Polypeptide through the Defined Twin-Arginine Translocation (Tat) Pathway

“…1A) and that YidC co- purifies with overproduced SecDFyajC, but not with overproduced SecYEG (Nouwen and Driessen, 2002). It has also been known that the YidC pathway has a threading mechanism for unfolded proteins (DeLisa et al, 2002), YidC can interact directly with a Sec-independent membrane protein (Chen et al, 2002), and E. coli YidC is essential for viability (Samuelson et al, 2000). These results demonstrated the presence of the novel Yid pathway (named for its association with the YidCrelated pathway), differing from the Sec pathway and possessing a SecDFyajC translocon.…”