The emergence and spread of chloroquine-resistant Plasmodium falciparum malaria parasites has been a disaster for world health. Resistance is conferred by mutations in the Chloroquine Resistance Transporter (PfCRT), an integral membrane protein localized to the parasite's internal digestive vacuole. These mutations result in a marked reduction in the accumulation of chloroquine (CQ) by the parasite. However, the mechanism by which this occurs is unclear. We expressed both wild-type and resistant forms of PfCRT at the surface of Xenopus laevis oocytes. The resistant form of PfCRT transported CQ, whereas the wild-type protein did not. CQ transport via the mutant PfCRT was inhibited by CQ analogs and by the resistance-reverser verapamil. Thus, CQ resistance is due to direct transport of the drug via mutant PfCRT.
Cyanobacteria possess a highly effective CO2-concentrating mechanism that elevates CO 2 concentrations around the primary carboxylase, Rubisco (ribulose-1,5-bisphosphate carboxylase͞oxy-genase). This CO 2-concentrating mechanism incorporates lightdependent, active uptake systems for CO 2 and HCO 3 ؊ . Through mutant studies in a coastal marine cyanobacterium, Synechococcus sp. strain PCC7002, we identified bicA as a gene that encodes a class of HCO 3 ؊ transporter with relatively low transport affinity, but high flux rate. BicA is widely represented in genomes of oceanic cyanobacteria and belongs to a large family of eukaryotic and prokaryotic transporters presently annotated as sulfate transporters or permeases in many bacteria (SulP family). Further gain-offunction experiments in the freshwater cyanobacterium Synechococcus PCC7942 revealed that bicA expression alone is sufficient to confer a Na ؉ -dependent, HCO 3 ؊ uptake activity. We identified and characterized three cyanobacterial BicA transporters in this manner, including one from the ecologically important oceanic strain, Synechococcus WH8102. This study presents functional data concerning prokaryotic members of the SulP transporter family and represents a previously uncharacterized transport function for the family. The discovery of BicA has significant implications for understanding the important contribution of oceanic strains of cyanobacteria to global CO 2 sequestration processes.SulP transporters ͉ CO2 sequestration ͉ photosynthesis I t is estimated that some 50% of global primary productivity occurs in the oceans, and marine cyanobacteria contribute significantly to this global CO 2 sequestration process (1). For example, in open oceans located between 40°N and 40°S, photosynthetic CO 2 fixation is dominated by marine cyanobacteria of the Synechococcus and Prochlorococcus genera, and together these species perform 30-80% of primary production (2, 3). The largest nutrient uptake flux encountered by marine cyanobacteria is for dissolved inorganic carbon (Ci), yet relatively little is known about the process of Ci accumulation for photosynthesis in the oceanic cyanobacteria, in contrast to our knowledge of freshwater strains.In aquatic systems, Ci exists mainly as two slowly interconvertible forms, CO 2 and HCO 3 Ϫ . In response to unique restrictions on the rate of CO 2 supply in the aquatic environment, cyanobacteria have evolved a very efficient mechanism for capturing CO 2 and HCO 3 Ϫ for photosynthetic fixation into sugars. The Ci-capturing mechanism functions as a CO 2 -concentrating mechanism because it effectively concentrates CO 2 around the main CO 2 -fixing enzyme, ribulose-1,5-bisphosphate carboxylase͞oxygenase (Rubisco). In the best characterized species, mostly freshwater cyanobacterial strains, the CO 2 -concentrating mechanism consists of several active uptake systems for CO 2 and HCO 3 Ϫ , plus a unique microcompartment called the carboxysome that contains the CO 2 -fixing enzyme, Rubisco (4-6).In recent years, the availability of a...
Crop yields need to nearly double over the next 35 years to keep pace with projected population growth. Improving photosynthesis, via a range of genetic engineering strategies, has been identified as a promising target for crop improvement with regard to increased photosynthetic yield and better water-use efficiency (WUE). One approach is based on integrating components of the highly efficient CO(2)-concentrating mechanism (CCM) present in cyanobacteria (blue-green algae) into the chloroplasts of key C(3) crop plants, particularly wheat and rice. Four progressive phases towards engineering components of the cyanobacterial CCM into C(3) species can be envisaged. The first phase (1a), and simplest, is to consider the transplantation of cyanobacterial bicarbonate transporters to C(3) chloroplasts, by host genomic expression and chloroplast targeting, to raise CO(2) levels in the chloroplast and provide a significant improvement in photosynthetic performance. Mathematical modelling indicates that improvements in photosynthesis as high as 28% could be achieved by introducing both of the single-gene, cyanobacterial bicarbonate transporters, known as BicA and SbtA, into C(3) plant chloroplasts. Part of the first phase (1b) includes the more challenging integration of a functional cyanobacterial carboxysome into the chloroplast by chloroplast genome transformation. The later three phases would be progressively more elaborate, taking longer to engineer other functional components of the cyanobacterial CCM into the chloroplast, and targeting photosynthetic and WUE efficiencies typical of C(4) photosynthesis. These later stages would include the addition of NDH-1-type CO(2) pumps and suppression of carbonic anhydrase and C(3) Rubisco in the chloroplast stroma. We include a score card for assessing the success of physiological modifications gained in phase 1a.
We report on the sequencing and analysis of a 3,557-bp genomic DNA clone that is located between 4.8 and 1.2 kilobase pairs (kb) upstream of the rbcL gene and is capable of complementing a class of cyanobacterium Synechococcus sp. strain PCC7942 mutants requiring a high level of CO2. The upstream 2,704 bp of this sequence is novel, the remaining 852 bp having been reported by other workers. Four new open reading frames (ORFs) have been identified along with putative promoter elements. These ORFs, which could code for proteins of 7, 10.9, 11, and 58 kDa in size, have been named ORF 64, ccmK, ccmL, and ccmM, respectively. The last three have been named ccm genes on the basis that insertional mutagenesis of each produces a phenotype requiring a high level of CO2 (i.e., each produces a lesion in the CO2 concentrating mechanism). The putative gene product for the large ccmM ORF has three internally repeated regions and also has two possible DNA binding motifs. Two defined mutants in the 3,557-bp region, mutants PVU and P-N, have been more fuly characterized. The PVU mutant has a drug marker inserted into the ccmL gene, and it possesses abnormal rod-shaped carboxysomes. The P-N mutant is a 2.64-kb deletion of DNA from the same position in ccmL to a region closer to rbcL. This mutant, which has previously been shown to lack carboxysomes and have soluble ribulosebiphosphate carboxylase/oxygenase activity, has now been shown to have a predominantly soluble carboxysomal carbonic anhydrase activity. Both mutants were found to possess carboxysomal carbonic anhydrase activities which are below wild-type levels, and in the P-N mutant this activity appears to be unstable. The results are discussed in terms of the possible interactions of putative ccm gene products in the process of carboxysome assembly and function.Cyanobacteria possess a CO2 concentrating mechanism (CCM) which functions to maintain an elevated CO2 level around the primary C02-fixing enzyme, ribulosebisphosphate carboxylase/oxygenase (Rubisco) (1). This results in Rubisco being close to substrate saturation for CO2. conferring to the cell the physiological and ecological benefits of an increase in photosynthetic performance. To date, two major components of the CCM have been identified. These are (i) a transport system which results in the accumulation of inorganic carbon (C1) within the cell and (ii) the Rubiscocontaining carboxysomes that function to provide a favorable environment for CO2 fixation (4).The basal form of the transport system is constitutively expressed, and although showing a clear preference for CO2 as the substrate, the active transporter will also accept HCO3 (4). Irrespective of the substrate, dissolved inorganic carbon (CJ) is accumulated inside the cell as HCO3f,
Ammonium is an important source of nitrogen for plants. It is taken up by plant cells via ammonium transporters in the plasma membrane and distributed to intracellular compartments such as chloroplasts, mitochondria and vacuoles probably via different transporters in each case. Ammonium is generally not used for long-distance transport of nitrogen within the plant. Instead, most of the ammonium transported into plant cells is assimilated locally via glutamine synthetases in the cytoplasm and plastids. Ammonium is also produced by plant cells during normal metabolism, and ammonium transporters enable it to be moved from intracellular sites of production to sites of consumption. Ammonium can be generated de novo from molecular nitrogen (N(2)) by nitrogen-fixing bacteria in some plant cells, such as rhizobia in legume root nodule cells, and at least one ammonium transporter is implicated in the transfer of ammonium from the bacteria to the plant cytoplasm. Plant physiologists have described many of these ammonium transport processes over the last few decades. However, the genes and proteins that underlie these processes have been isolated and studied only recently. In this review, we consider in detail the molecular structure, function and regulation of plant ammonium transporters. We also attempt to reconcile recent discoveries at the molecular level with our knowledge of ammonium transport at the whole plant level.
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