Glutamine Transport and the Role of the Glutamine Translocator in Chloroplasts

The transport of L-['4Cqglutamine in oat (Avena sativa L.) and spinach (Spinacia oleracea L.) chloroplasts was studied by a conventional single-layer and a newly developed stable double-layer silicone oil filtering system. I"CiGlutamine was actively transported into oat chloroplasts against a concentration gradient. Metabolite uptake was greatly affected by the endogenous dicarboxylate pools, which could be easily changed by preloading the chloroplast with specific exogenous substrate. Gluta- mine uptake was decreased by 44 to 75% in oat chloroplasts preloaded with malate, 2-oxoglutarate (2-OG), and aspartate, but increased by 52% in chloroplasts preloaded with L-glutamate. On the other hand, the uptake of the other four dicarboxylates was decreased by 47 to 79% in chloro- plasts preloaded with glutamine. In glutamine-preloaded chloroplasts the uptake of glutamine was inhibited only by L-glutamate. The observed inhibition by L-glutamate was competitive with an apparent Ki value of 32.1 millimolar in oat and 6.7 millimolar in spinach chloroplasts. This study indicates that there are two components involved in glutamine transport in chloroplasts. The major component was mediated via a specific glutamine translocator. It was specific for glutamine and did not transport other dicarboxylates except L-glutamate. A Ko.5 value of 1.25 millimolar and V.,, of 45.5 micromoles per milligram of chlorophyll per hour were determined for the glutamine translocator in oat chloroplasts. The respective values were 1.0 millimolar and 16.7 micromoles per milligram of chlorophyll per hour in spinach chloroplasts. A three-translocator model, involving the glutamine, dicarboxylate, and 2-OG translocators, is proposed for the reassimilation of photorespiratory NH3 in chloroplasts of C3 species. In this three-translocator model the addi-tional transport of glutamine into the chloroplast is coupled to the export of glutamate via the glutamine translocator. This is an extension of the two-translocator model, involving the dicarboxylate and 2-OG translocators, proposed for spinach chloroplasts, (KC Woo, UI Flugge, HW Heldt 1987 Plant Physiol 84: 624-632). These results indicate the presence of a distinct site for gluta- mine transport in these chloroplasts. Furthermore glutamate, but not aspartate (or malate or 2-OG), could apparently also be transported via this glutamine translocator. Interestingly the

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