Department of Physics, Chemistry and Biology (IFM)

The amino-acid sequence give hints about how the protein looks like which in-turn gives us information about the alignment and function. There are a number of trans-membrane domains and a Poly-G which assumes a U-conformation⁵⁴. This gives us an idea about the positioning of ANTR3 in the plastid membrane. The hydophobicity or hydrophilicity of amino acids provide an understanding of the arrangement of amino acids in the cell which in-turn would provide details regarding function, structure and localization. These initial predictions and understanding would help us a lot in further protein studies.

The predicted molecular weight of the ANTR3 is 55 KDa but when these are loaded into wells in a SDS - PAGE gel and blotted on to a PVDF membrane, bands are viewed at 45 KDa which differs significantly from its theoretical size. Similar migration shift was observed for ANTR1 and ANTR2³⁷. One possibility can be incorrect prediction of cleavage site. Since all three proteins show a difference from its theoretical molecular weight, the possible explanation is the presence of charged and hydrophilic residues³⁷.   

All members of this family have been expressed heterologously in yeast and E. coli and all of them had an optimum pH of 5 except one member whose optimum value was 7.5³⁷. In this study, we have focussed on the protein in-vivo where mutants were taken advantage of. Moreover, it is impossible to study this protein without mutants because of the presence of other transporters. Highest activity has been observed at pH 7.5 in presence of NaCl. Transport experiments were done in presence of inhibitors to confirm that we are studying phosphate transport. Mersalyl disrupts S-S bridges in the transporter and it had considerable effects on the transport activity. At this present stage we know there are cystine residues but there is no knowledge about the formation of these bonds. PLP is a well known phosphate transporter inhibitor since it is an analogue of phosphate and blocks transport while FCCP affects the electrochemical gradient. These inhibitors had pronounced effect on the activity so we confirm that it was phosphate transport that we were studying.

Back-exchange was done with the same pH of 6.5+ Na to 6.5+ Na to check out leakage of Pi from plastids by any other sources like other transporters. The uptake with inhibitors was performed at pH 6.5+ Na for the wild type and 7.5+ Na for wild type and mutants. This is because of the fact that plastids show the best transport activity under a pH of 7.5 in presence of cations.

As per our results, mutants were found to grow 30 % more profusely than the wild-type. At the same time starch levels in the roots of mutants were reduced, which is consistent with the idea that phosphate export from a root plastid would lead to increased phosphate levels and that this would inhibit starch synthesis (unpublished data, Wayne Versaw, Univ. Of Texas ). Our transport assays in intact plastids strongly supports this argument. Repeated starch and sugar measurements in leaves, roots and phloem exudates with the idea that source / sink allocation may be broadly affected by mutation. Glucose and sucrose levels in roots showed differences in the mutant but more samples are needed to make a marked difference. Amino acid content was unaffected so it is not a matter of total carbon or nitrogen that is out of balance (unpublished data, Wayne Versaw, University of Texas ).

Previous studies have recorded transcript levels by using RT-PCT (reverse transcriptase- PCR ) and ANTR3 transcript levels were found to be decreased by 80 % during light treatment unlike other protein of the same family²⁹. Moreover these transcript levels were more abundant in dark rather than in light and not under circadian control²⁹.