BIOCHEMICAL STUDIES OF ATP-DRIVEN BACTERIAL TRANSPORT

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Kdp is an ATP-driven potassium transport system of Escherichia coli. It consists of three proteins, KdpA, KdpB and KdpC, encoded by the three genes of the kdpABC operon. Expression of the kdpABC operon is under the control of the two products of the adjacent kdpDE operon. Kdp is functionally similar to transport ATPases with an acyl-phosphate intermediate. The sequence of KdpB is homologous to the Ca-ATPase of sarcoplasmic reticulum. Major emphasis remains on genetic approaches: 1. Clone kdpABC operon behind a strong, controllable promoter. 2. Isolate, characterize and sequence mutations which alter but do not abolish Kdp function. Mutations which reduce affinity for K are under study; many more such mutations will be investigated. Mutations whose primary effect is to reduce the Vmax of transport will also be studied. 3. KdpA has been identified as having a major role in forming the K binding site of Kdp. The sequence of KdpA suggests it crosses the membrane many times. Topological will be performed to identify which parts of KdpA are exposed internally, which are exposed externally and which are in the membrane. 4. Determine subunit interactions in the functional Kdp complex. 5. Identify and characterize the products of the kdpDE operon. 6. Characterize the kdpABC promoter: sequence the promoter region and determine the site of transcription initiation. 7. Clone kdpDE behind a strong, controllable promoter. 8. Study properties of the proteins encoded by the kdpDE operon; attempt to define their roles in regulation. This project has beniffted from collaboration with Karlheinz Altendorf (Univ. of O nabruck, West Germany) for several years. This collaboration continues; work to be done mainly or exclusively in Osnarbruck, includes 9. Purification to homogeneity of Kdp. 10. Determination of the N-terminal sequence of KdpB; determination of the C-terminal residues of KdpA, KdpB and KdpC.