YEAST MITOCHONDRIAL PHOSPHATE-TRANSPORT PROTEIN EXPRESSED IN ESCHERICHIA-COLI - SITE-DIRECTED MUTATIONS AT THREONINE-43 AND AT A SIMILAR LOCATION IN THE 2ND TANDEM REPEAT (ISOLEUCINE-141)

Yeast mitochondrial phosphate transport activity has been reconstitute d from the import receptor (MIR) expressed as inclusion bodies in Esch erichia coli. This result undermines the suggestion [Murakami, H., et al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 3358-3362] that the MIR h as been misidentified as the phosphate transport protein (PTP). PTP wa s solubilized with N-lauroylsarcosinate and Triton X-100 and purified with a yield of about 2 mg/L of induced bacterial culture. This PTP, r econstituted in liposomes, catalyzes phosphate uptake with a V-max [24 .5 degrees C, net (zero trans), pH(i) = 8.0, pH(e) = 6.8] of 0.61 mmol of phosphate min(-1) (mg of PTP)(-1) and a K-m of 1.30 mM. This V-max is higher and the K-m about the same as that obtained with PTP purifi ed from mitochondria. Replacement of Thr43 and Ile141 by other amino a cids results in three types of PTP: (a) 2.5-5.0% V-max of wild-type PT P (PTPwt) (Thr43Cys; Thr43Ser; Ile141Cys), (b) <0.1% V-max (detection limit of assay) of PTPwt (Thr43Ala; Thr43Asp), and (c) proton transpor t uncoupled from phosphate transport (Ile141Cys). K-m changes are not significant. Activity of Thr43Cys confirms results obtained with mitoc hondrially expressed protein. Thus, yeast PTP requires Thr43 and mamma lian PTP the similarly located Cys42 for high transport activity. Thr4 3 and Ile141 are each situated between two basic residues (LysThrArg v s ArgIleArg). Cys substitutions in either of these positions confer th e same high N-ethylmaleimide sensitivity to the yeast PTPwt as display ed by the mammalian PTP. On the basis of single substitutions and a ba cteriorhodopsin-like pattern of Glu126, His32, and Glu137, helices I a nd III (second tandem repeat) have been suggested to facilitate proton transport. Thus it is interesting that replacing Ile141, which is loc ated near the matrix end of transmembrane helix III (similar to Thr43 and helix I), with a polar Cys perturbs helix III and its local enviro nment sufficiently to yield uncoupled proton transport.