Mitochondrial Phosphate Transport Protein. Reversions of Inhibitory Conservative Mutations Identify Four Helices and a Nonhelix Protein Segment with Transmembrane Interactions and Asp39, Glu137, and Ser158 as Nonessential for Transport

Abstract

The mitochondrial phosphate transport protein (PTP) has six (A--F) transmembrane (TM) helices per subunit of functional homodimer with all mutations referring to the subunit of the homodimer. In earlier studies, conservative replacements of several residues located either at the matrix end (Asp39/helix A, Glu137/helix C, Asp236/helix E) or at the membrane center (His32/helix A, Glu136/helix C) of TM helices yielded inactive single mutation PTPs. Some of these residues were suggested to act as phosphate ligands or as part of the proton cotransport path. We now show that the mutation Ser158Thr, not part of a TM helix but located near the center of the matrix loop (Ile141--Ser171) between TM helices C and D, inactivates PTP and is thus also functionally relevant. On the other side of the membrane, the single mutation Glu192Asp at the intermembrane space end of TM helix D yields a PTP with 33% wild-type activity. We constructed double mutants by adding this mutation to the six transport-inactivating mutations. Transport was detected only in those with Asp39Asn, Glu137Gln, or Ser158Thr. We conclude that TM helix D can interact with TM helices A and C and matrix loop Ile141--Ser171 and that Asp39, Glu137, and Ser158 are not essential for phosphate transport. Since our results are consistent with residues present in all 12 functionally identified members of the mitochondrial transport protein (MTP) family, they lead to a general rule that specifies MTP residue types at 7 separate locations. The conformations of all the double mutation PTPs (except that with the matrix loop Ser158Thr) are significantly different from those of the single mutation PTPs, as indicated by their very low liposome incorporation efficiency and their requirement for less detergent (Triton X-100) to stay in solution. These dramatic conformational differences also suggest an interaction between TM helices D and E. The results are discussed in terms of TM helix movements and changes in the PTP monomer/dimer ratio.

Single Replacement Constructs of All Hydroxyl, Basic, and Acidic Amino Acids Identify New Function and Structure-Sensitive Regions of the Mitochondrial Phosphate Transport Protein

“…Transport ActiVity of Mutant PTPs. Figure 1 shows the results of the transport assays of all the new mutants as well as relevant mutants from earlier studies (8,10,11). All of the data reflect the results of at least two initial rate transport assays using the same PTP proteoliposome preparation.…”

“…Figure 2 shows a diagram of the most recent PTP model (10). It indicates those residues that upon replacement catalyze P i uptake at <0.6 µmol of P i min -1 (mg of PTP) -1 (heavy circles with single letter code amino acid) and those with uptake rates of >0.6 but <6.0 µmol of P i min -1 (mg of PTP) -1 (light circles with single letter code amino acid).…”

“…Expression of Mutant PTPs. The yeast PTP gene (9) in the plasmid pNYns (10) was expressed in Escherichia coli BL21(DE3) cells (Novagen). The transformed E. coli cells were grown to an OD (600 nm) of 0.8, IPTG was added to 1.0 mM, and the cells were grown for another 3 h. The cells were harvested and the pellets stored at -20 °C.…”

Redundancy in the function of mitochondrial phosphate transport in Saccharomyces cerevisiae and Arabidopsis thaliana

“…In S. cerevisiae , a defect in phosphate transport impairs growth on non‐fermentable substrates and is associated with a reduction in the membrane potential and consequently with an inhibition of mitochondrial protein import (Zara et al ., 1996). Site‐directed mutagenesis experiments identified residues of Mir1p that are important for the structure and phosphate/proton transport activity (Briggs et al ., 1999; Phelps et al ., 2001; Wohlrab et al ., 2002 and see Supplementary material Fig. S1).…”

SIPP, a Novel Mitochondrial Phosphate Carrier, Mediates in Self-Incompatibility

“…The amino acids known to be required for phosphate transport in Mir1 are . When these residues are mutated, the ability to transport phosphate is suppressed (Briggs et al, 1999;Phelps et al, 2001;Wohlrab et al, 2002). Sequence alignment of NaSIPP with multiple functional and putative MPCs showed high conservation in these 12 residues for NaSIPP (Supplemental Fig.…”