The essential role of YidC in bacterial membrane protein insertion

The objective of this research was to provide a greater understanding of membrane protein biogenesis by studying this process in bacteria. The basic machinery for the translocation of proteins into or across membranes is remarkably conserved from Escherichia coli to humans. In eukaryotes, proteins are inserted into the endoplasmic reticulurn (ER) by the Sec61 trimeric complex. In bacteria, the homologous SecYEG complex forms a translocation channel across the plasma membrane. In addition to serving as the primary protein export apparatus in bacteria, the Sec-translocase mediates the membrane insertion of most proteins studied. Since some membrane proteins do not exhibit dependence on the Sec-translocase, it was proposed that a novel machinery may function specifically for membrane protein integration. Evidence for such a machinery was discovered by studies of yeast mitochondria. The postulated bacterial ancestry of mitochondria suggests that the process of membrane protein insertion should be evolutionarily conserved. However, mitochondria do not contain the Sec components. Instead, Oxa1p was shown to mediate the insertion of a subset of proteins into the inner membrane from the mitochondrial matrix. Furthermore, the Oxa1p machinery was shown to mediate amino-terminal translocation of membrane proteins, a process not accounted for by the Sec model of membrane insertion. An OXA1 homology search revealed counterparts in the chloroplast and in bacteria suggesting the possibility of a conserved Sec-independent pathway for membrane insertion. A search of the E. coli genome identified the uncharacterized yidC gene. The yidC gene encodes a 60 kDa membrane protein with a topology similar to the topology of Oxa1p.; Attempts were made to delete yidC in E. coli in order to study the physiological role of the gene product. These attempts were unsuccessful, which provided indirect evidence that yidC is essential for viability. A YidC-depletion strain was constructed where yidC expression is under control of the araBAD operator/promoter. Upon isolation, a streak of this strain on nutrient agar containing glucose to repress expression of yidC resulted in no growth after 16 hours at 37°C, confirming that yidC is essential for bacterial viability. In this strain, designated JS7131, the level of YidC is depleted by growth in liquid media containing glucose and any effects on membrane protein insertion can be assayed.; In vivo assays using JS7131 determined that the Sec-independent proteins M13 procoat and Pf3 coat absolutely require YidC for insertion into the inner membrane. In addition, amino-terminal translocation of Pf3-Lep is severely affected by YidC depletion. These results suggested that YidC is a primary component of a novel translocation machinery distinct from the Sec complex. However, depletion of YidC also affects the membrane insertion of Sec-dependent proteins so it appears that YidC may also cooperate with the Sec-translocase. The Sec-mediated export of proteins to the periplasm, or the outer membrane is only slightly delayed in YidC-depleted cells. Therefore, YidC is a component of the machinery specialized for the integration of membrane proteins.