Abstract
The YidC/Oxa1/Alb3 family of proteins catalyzes membrane protein insertion in bacteria, mitochondria, and chloroplasts. In this study, we investigated which regions of the bacterial YidC protein are important for its function in membrane protein biogenesis. In Escherichia coli, YidC spans the membrane six times, with a large 319-residue periplasmic domain following the first transmembrane domain. We found that this large periplasmic domain is not required for YidC function and that the residues in the exposed hydrophilic loops or C-terminal tail are not critical for YidC activity. Rather, the five C-terminal transmembrane segments that contain the three consensus sequences in the YidC/Oxa1/Alb3 family are important for its function. However, by systematically replacing all the residues in transmembrane segment (TM) 2, TM3, and TM6 with serine and by swapping TM4 and TM5 with unrelated transmembrane segments, we show that the precise sequence of these transmembrane regions is not essential for in vivo YidC activity. Single serine mutations in TM2, TM3, and TM6 impaired the membrane insertion of the Sec-independent procoat-leader peptidase protein. We propose that the five C-terminal transmembrane segments of YidC function as a platform for the translocating substrate protein to support its insertion into the membrane.
Highlights
In prokaryotes and eukaryotes, the YidC/Oxa1/Alb3 family of proteins mediates membrane protein insertion
We found that the large N-terminal periplasmic domain, periplasmic loops, and cytoplasmic regions of YidC are not directly involved in function, but that the five C-terminal transmembrane regions play an important role
We propose that the C-terminal transmembrane segments of YidC function as a platform or framework in membrane protein insertion
Summary
The five C-terminal transmembrane segments that contain the three consensus sequences in the YidC/ Oxa1/Alb family are important for its function. Unlike Oxa and Alb, which span the membrane five times with an N-terminal luminal tail of 100 residues or less [19], YidC spans the membrane six times and, following the first transmembrane region, has a large N-terminal domain of 319 residues in the periplasm (see Fig. 1) [20]. We found that the large N-terminal periplasmic domain, periplasmic loops, and cytoplasmic regions of YidC are not directly involved in function, but that the five C-terminal transmembrane regions play an important role This important role of the five transmembrane regions is consistent with sequence alignment analysis of the Oxa family members, which almost all have five transmembrane segments [21]. We propose that the C-terminal transmembrane segments of YidC function as a platform or framework in membrane protein insertion
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