The essential role of YidC in bacterial membrane protein insertion

The objective o f 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 reticulum (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 o f 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 o f membrane protein insertion should be evolutionarily conserved. However, mitochondria do not contain the Sec components. Instead, Oxa I p was shown to mediate the insertion of a subset o f proteins into the inner membrane from the mitochondrial matrix. Furthermore, the Oxa Ip machinery was shown to mediate amino-terminal translocation of membrane proteins, a process not accounted for by the Sec model o f membrane insertion. An OXAl homology search revealed counterparts in the chloropiast and in bacteria indicating the possibility o f a conserved Sec-independent pathway for membrane insertion. A search of the E. coli genome identified the uncharacterized y/dC gene TheyidC gene encodes a 60 kDa membrane protein with a topology similar to the topology of Oxa Ip. Attempts were made to delete 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 _y/V/C expression is under control o f the aroBAD operator/promoter. Upon isolation, a streak of this strain on nutrient agar containing glucose to repress expression oïyidC resulted in no growth after 16 hours at 37°C, confirming xhaXyidC 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 efiects on membrane protein insertion can be assayed. In vivo assays using JS7I31 determined that the Sec-independent protein, MI3 procoat, absolutely requires YidC for insertion into the inner membrane. In addition, amino-terminal translocation o f Pf3-Lep is severely affected by YidC depletion. These two results suggest 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 o f proteins to the periplasm or the outer membrane is only slightly delayed inYidC-depleted cells. Therefore, YidC is a component of the machinery specialized for the integration of membrane proteins.