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Abstract
A detailed comparison of citrate uptake into the vacuole-like lutoids of rubber tree (Hevea brasiliensis Muell. Arg.) and of malate and citrate transport into barley (Hordeum vulgare L.) vacuoles revealed very similar transport specificities. In order to identify proteins mediating the transport, two photoreactive analogues (N'-(2-hydroxy-5-azido)-diazo-N-3,5-benzenedicarboxylic acid and 5-azidoisophthalic acid) of malate/citrate were synthesized and found to efficiently inhibit citrate uptake into barley vacuoles (Ki = 18 microM) and Hevea lutoid vesicles (Ki = 27 microM). In vacuoles from both plant species, these photoaffinity probes specifically labeled a single protein with a molecular mass of 23.6 kDa. This citrate binding protein (CBP) was purified to homogeneity from Hevea lutoids, and amino acid sequences were determined for NH2-terminal and tryptic peptides. Using degenerate oligonucleotides of the NH2-terminal sequence, a cDNA coding for the CBP protein of Hevea was isolated. The cDNA codes for a precursor protein of 238 amino acids, containing an NH2-terminal 31-amino acid signal sequence for endoplasmic reticulum targeting, a prerequisite for vacuolar localization. The mature CBP does not show significant sequence similarities to any known primary protein structure and thus represents a member of a novel class of proteins.
Highlights
A detailed comparison of citrate uptake into the vacuole-like lutoids of rubber tree
A detailed comparison of the uptake of citrate and malate into barley vacuoles showed that the characteristics of uptake are very similar and this suggested that malate and citrate are transported by the same transport system across barley tonoplasts [14]
We have compared citrate uptake into barley vacuoles and Hevea lutoid vesicles and were able to show that transport is efficiently inhibited by two photoreactive dicarboxylates
Summary
A detailed comparison of citrate uptake into the vacuole-like lutoids of rubber tree In order to identify proteins mediating the transport, two photoreactive analogues (N-(2-hydroxy-5-azido)-diazo-N-3,5-benzenedicarboxylic acid and 5-azidoisophthalic acid) of malate/citrate were synthesized and found to efficiently inhibit citrate uptake into barley vacuoles (Ki ؍18 M) and Hevea lutoid vesicles (Ki ؍27 M). We have compared citrate uptake into barley vacuoles and Hevea lutoid vesicles and were able to show that transport is efficiently inhibited by two photoreactive dicarboxylates. These photoaffinity probes label a 23.6kDa protein in both plant systems. We describe the isolation of the citrate binding protein of Hevea, the isolation of the corresponding cDNA and its gene, as well as the identification of homologous genes in barley
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