paper4

Palmieri et.  al.
          David Bruce
       


    The purpose of the research described in this paper was to characterize the nature of the mitochondrial aspartate/glutamate carrier (AGC).  Previous experiments had identified the existence of such a carrier and had established the electrogenic aspect of its function; it had been hypothesized that this was a protein carrier, and this research conclusively identifies the proteins citrin and aralar1 as functioning as the AGC in mitochondria.  Because mutations in the citrin gene have previously been shown to result in type II citrullinemia, an inherited metabolic defect, the authors through this work also manage to postulate a reasonable explanation of the biochemical etiology of this disorder.
    Although the exact proteins involved in the AGC had not been identified prior to this research, much was already known about the AGC.  Because the mitochondrial inner membrane is mostly impermeable to free diffusion (with the exception of water, oxygen, and carbon dioxide), it is apparent that most substrates entering or leaving the mitochondria must experience some sort of facilitated transport.  Other transport proteins had been characterized; further, experiments in which mitochondrial systems are exposed to aspartate and/or glutamate and responses are measured via assay for efflux and influx as compared to the original state can establish a correlation between aspartate and glutamate -related behavior without precisely identifying what protein(s) are involved.  Experiments which test the function of mitochondria in response to different combinations of various substrates have been important.  Over thirty years of research had been done on the AGC before the proteins involved were actually identified.
    It appears that the authors, through assiduous study of past literature and careful thought, identified citrin and aralar1 as candidates for the AGC system.  As the genes encoding these proteins were known, E.  Coli transformants which were designed to overexpress either the citrin or the aralar1 gene were created; transformants designed to overexpress just the C-terminal domain of each protein were also developed.  The expressed protein products were then isolated and identified using various techniques including N-terminal sequencing to ensure the identity and purity of the product.
    These protein isolates were then reconstituted into liposomes.  By utilizing varying concentrations of a multitude of internal and external substrates, they showed fairly conclusively that citrin and aralar1 were specific transporters of aspartate and glutamate; unitransport was not observed, indicating that exchange was important.  The authors further noted that by inducing a K+ diffusion potential across the liposomal membrane, aspartate intake was decreased, output was increased, and the reverse effect was noticed for glutamate.  This effect was not observed in the absence of a diffusion potential; the electrogenic nature of these exchanges added to the evidence that citrin and aralar1 were the AGC transport proteins.  Finally, when HEK-293T cells were modified to overexpress aralar1 and citrin, permeabilized with digitonin, and incubated with a potent brew consisting of glutamate, malate, and lactate, the reduction of MTT was significantly greater in the overexpressing cells as opposed to control.  Because overexpression of the AGC would be expected to lead to increased reduction of MTT, the authors conclude that this constitutes good evidence that aralar1 and citrin are in fact the AGC carriers.

    The domain structures of citrin and aralar1 are quite similar.  Both contain four EF-hand Ca2+ binding motifs in their N-terminal domain; and also contain C-terminal domains which are fairly homologous to structures seen in known mitochondrial transporters.  What this structure suggests is (1) that these proteins may be involved in mitochondrial transport and (2) that this transport may be regulated by Ca2+ interactions.
    Figure 5 in this paper presents a complex series of data intended to demonstrate the external Ca2+ dependence of the AGC.  Figure 5(A) is a diagram of the various pathways involved in glutamate transport and metabolism in the mitochondria; substrates involved in cytosolic reactions involving malate, alpha-ketoglutarate, oxaloacetate, aspartate (and their interactions which produce NADH) are also depicted as well as the calcium transport system, the AGC, etc.  Essentially what the authors are doing, as described in the figure, is comparing what happens when pathways are manipulated and transporters (i.e. the calcium transporter) are blocked so that cellular CO2 production accurately reflects the contribution of the AGC alone to glutamate decarboxylation within the mitochondria.  They note that when ruthenium red is added (which blocks Ca2+ transport into the mitochondria) in the absence of AOAA (which inhibits aspartate transferase, and thus blocks the AGC pathway for glutamate decarboxylation) but the presence of cytosolic CaCl2, no inhibitory effect is seen; thus, they conclude that the external Ca2+ concentration has a stimulatory effect, since the internal transport of Ca2+ is blocked in this case.
    The authors speculate that external Ca2+ control of the AGC is related to the fact that, as opposed to intramitochondrial Ca2+-stimulated reactions, the AGC system is not close to equilibrium, and that external Ca2+ stimulation enables the AGC transporters to increase the supply of reducing equivalents to the mitochondria without a net consumption of metabolites.
    Type II citrullinemia is typified by the liver-specific deficiency of argininosuccinate synthase (ASS), which catalyzes the synthesis of argininosuccinate from citrulline and aspartate.  This syndrome takes place in the absence of any deformation in either the ASS mRNA or the ASS gene itself.  The known defect in citrin, which causes this disorder, would lead to an impairment in the AGC, disturbing the function of the malate/aspartate shuttle , and leaving a deficit in cytosolic aspartate for the synthesis of argininosuccinate.  Why this should lead to diminished ASS activity in the liver is unknown, but the authors postulate that possibly a feed-forward loop required to activate the enzyme is disrupted.