THE MEMBRANE TOPOLOGY OF THE RHIZOBIUM-MELILOTI C4-DICARBOXYLATE PERMEASE (DCTA) AS DERIVED FROM PROTEIN FUSIONS WITH ESCHERICHIA-COLI K12 ALKALINE-PHOSPHATASE (PHOA) AND BETA-GALACTOSIDASE (LACZ)

The Rhizobium meliloti dctA gene encodes the C4-dicarboxylate permease which mediates uptake of C4-dicarboxylates, both in free-living and s ymbiotic cells. Based on the hydrophobicity of the DctA protein, 12 pu tative membrane spanning regions were predicted. The membrane topology was further analysed by isolating in vivo fusions of DctA to Escheric hia coli alkaline phosphatase (PhoA) and E. coli beta-galactosidase (L acZ). Of 10 different fusions 7 indicated a periplasmic and 3 a cytopl asmic location of the corresponding region of the DctA protein. From t hese data a two-dimensional model of DctA was constructed which compri sed twelve transmembrane alpha-helices with the amino-terminus and the carboxy-terminus located in the cytoplasm. In addition, four conserve d amino acid motifs present in many eukaryotic and prokaryotic transpo rt proteins were observed.