The sensor protein KdpD inserts into the Escherichia coli membrane independent of the Sec translocase and YidC

Abstract

KdpD is a sensor kinase protein in the inner membrane of Escherichia coli containing four transmembrane regions. The periplasmic loops connecting the transmembrane regions are intriguingly short and protease mapping allowed us to only follow the translocation of the second periplasmic loop. The results show that neither the Sec translocase nor the YidC protein are required for membrane insertion of the second loop of KdpD. To study the translocation of the first periplasmic loop a short HA epitope tag was genetically introduced into this region. The results show that also the first loop was translocated independently of YidC and the Sec translocase. We conclude that KdpD resembles a new class of membrane proteins that insert into the membrane without enzymatic assistance by the known translocases.When the second periplasmic loop was extended by an epitope tag to 27 amino acid residues, the membrane insertion of this loop of KdpD depended on SecE and YidC. To test whether the two periplasmic regions are translocated independently of each other, the KdpD protein was split between helix 2 and 3 into two approximately equal-sized fragments. Both constructed fragments, which contained KdpD-N (residues 1-448 of KdpD) and the KdpD-C (residues 444-894 of KdpD), readily inserted into the membrane. Similar to the epitope-tagged KdpD protein, only KdpD-C depended on the presence of the Sec translocase and YidC. This confirms that the four transmembrane helices of KdpD are inserted pairwise, each translocation event involving two transmembrane helices and a periplasmic loop.

The Cytoplasmic C-Terminal Domain of the Escherichia coli KdpD Protein Functions as a K + Sensor

“…Restriction analysis and DNA sequencing confirmed the introduction of the new restriction sites. Plasmids encoding KdpD-N (i.e., encoding residues 1 to 448 of KdpD) and KdpD-C (i.e., encoding residues of 444 to 894 of KdpD) have been described previously (7). To construct plasmids pSF1 and pSF2, the sequences coding for KdpD-N and KdpD-C were cleaved with XbaI/ HindIII and cloned into pBAD33 and pBAD18 (8), respectively.…”

“…KdpD was split into halves between helix 2 and 3 to generate fragments KdpD-N and KdpD-C. These fragments were expressed independently and were both found to insert into the cytoplasmic membrane (7). When plasmids encoding wild-type KdpD, KdpD-N, or KdpD-C were introduced into the kdpD deletion strain E. coli TKV2208, the N-terminal fragment (KdpD-N) alone did not allow the cells to grow at low K ϩ concentrations (0.1 mM).…”

Reversible Topological Organization within a Polytopic Membrane Protein Is Governed by a Change in Membrane Phospholipid Composition

“…Formation of a helical hairpin may sufficiently increase the net hydrophobicity of the sequence to drive polar loop residues across the lipid bilayer. Pairing of two transmembrane segments in the E. coli tetracycline antiporter TetA(C) and sensor kinase protein KdpD was suggested to be essential for integration into the membrane (49,50). The N-terminal hairpin of PheP appears to behave as an independent and very mobile insertion and translocation unit.…”

The role of lipids in membrane insertion and translocation of bacterial proteins

“…Also the polytopic melbiose permease, MelB, and recently the sensor kinase protein KdpD were found to insert into the membrane independent of Sec proteins, and in the case of KdpD also independent of YidC [123,124]. These Sec-independent proteins have a common topology with their N-and C-termini in the cytosol and all contain very small periplasmic loops.…”