Abstract
We previously constructed a bifunctionally active membrane-bound fusion protein, in which Escherichia coli proline carrier (the product of the putP gene) was linked with beta-galactosidase (the product of the lacZ gene) through a collagen linker (Hanada, K., Yamato, I., and Anraku, Y. (1987) J. Biol. Chem. 262, 14100-14104). The proline carrier was purified from this site specifically cleavable fusion protein. Cytoplasmic membranes overproducing the fusion protein were solubilized with dodecylmaltoside, and the solubilized fraction was subjected to anti-beta-galactosidase IgG-Sepharose chromatography. The fusion protein was specifically adsorbed to the immunoaffinity resin and then treated with collagenase for splitting the proline carrier moiety of the fusion protein from the beta-galactosidase moiety. The collagenase used for the collagenolysis was then removed by anti-collagenase IgG-Sepharose chromatography. In this way, the proline carrier was purified to more than 95% homogeneity of the protein. Proline transport in proteoliposomes reconstituted with the purified carrier was dependent on the membrane potential and the chemical gradient of Na+ across the membrane with apparent Michaelis constants for proline and for Na+ stimulation of 3.6 microM and 31 microM, respectively. These results indicated that the proline carrier mediates electrogenic Na+/proline symport.
Highlights
14104).The proline carriewras purified fromthis site the proline transport mechanism in a molecular level
These results indicated that the proline tiation protein of plasmid R6Kwas isolated by the carrier mediates electrogenic Na’/proline symport. elegant technique of site-specific proteolysis at a collagen linker inserted between the two moieties in thefusion protein
Thenucleotide sequence of the putP gene was determined, and, from the sequence, the carrier was predicted to consist of 502 amino acid residues, with an M, of 54,343 and to be a very h y ~ ophobi cprotein with 12 membrane-spanning segments [3].The proline carrier was identified as a cytoplasmic membrane protein, and its apparent M, of which on SDS-polyacrylamide gel electrophoresis was found to depend on the gel concentration [4]
Summary
The filter was washed once with 6 mlof0.1 M LiC1, and the radioactivity trapped on the filter was measured as described previously [11].The initial rate of the transportwas determined at theinitial 30-s point of incubation. Miscellurteous Methods-The proline carrier moiety in membrane vesicles was labeled with MalN[3H]Et as described elsewhere [4, 21]. Accurate determination of the protein of purified carrier in proteoliposomes was difficult because of the presence of a large excess of phospholipid. In reconstitution of the purified carrier, 80% of the carrier molecules were recovered in proteoliposomes, judging from the recoveryof radioactivity of MalN[3H]Et-labeled carrier.' So, the protein of purified carrier in proteoliposomeswas estimated to be 80% of that used in the reconstitution step
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