The structural and functional units of heteromeric amino acid transporters - The heavy subunit rBAT dictates oligomerization of the heteromeric amino acid transporters
ABSTRACT Heteromeric amino acid transporters are composed of a catalytic light subunit and a heavy subunit linked by a disulfide bridge. We analyzed the structural and functional units of systems b0,+ and xC-, formed by the heterodimers b0,+ AT-rBAT and xCT-4F2hc, respectively. Blue Native gel electrophoresis, cross-linking, and fluorescence resonance energy transfer in vivo indicate that system b0,+ is a heterotetramer [b0,+ AT-rBAT]2, whereas xCT-4F2hc seems not to stably or efficiently oligomerize. However, substitution of the heavy subunit 4F2hc for rBAT was sufficient to form a heterotetrameric [xCT-rBAT]2 structure. The functional expression of concatamers of two light subunits (which differ only in their sensitivity to inactivation by a sulfhydryl reagent) suggests that a single heterodimer is the functional unit of systems b0,+ and xC-.
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ABSTRACT: The x(c) (-) cystine/glutamate antiporter is a major plasma membrane transporter for the cellular uptake of cystine in exchange for intracellular glutamate. Its main functions in the body are mediation of cellular cystine uptake for synthesis of glutathione essential for cellular protection from oxidative stress and maintenance of a cystine:cysteine redox balance in the extracellular compartment. In the past decade it has become evident that the x(c) (-) transporter plays an important role in various aspects of cancer, including: (i) growth and progression of cancers that have a critical growth requirement for extracellular cystine/cysteine, (ii) glutathione-based drug resistance, (iii) excitotoxicity due to excessive release of glutamate, and (iv) uptake of herpesvirus 8, a causative agent of Kaposi's sarcoma. The x(c) (-) transporter also plays a role in certain CNS and eye diseases. This review focuses on the expression and function of the x(c) (-) transporter in cells and tissues with particular emphasis on its role in disease pathogenesis. The potential use of x(c) (-) inhibitors (e.g., sulfasalazine) for arresting tumor growth and/or sensitizing cancers is discussed.Journal of Cellular Physiology 06/2008; 215(3):593-602. DOI:10.1002/jcp.21366 · 3.87 Impact Factor
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ABSTRACT: The frog Xenopus laevis is an excellent model system to analyze the molecular processes involved in early embryogenesis and organogenesis. The advantages include a large number of embryos and the development that takes place outside the maternal body allowing for a continuous observation of morphogenetic events in the highly transparent larvae. In this work the function of the tissue specific transcription factor HNF1β was examined particularly in kidney development of Xenopus laevis. For this purpose it was made use of the binary Cre/loxP-system with activator and effector strains. In this system the Cre recombinase can be conditionally activated by a heat-shock. Transcription of the cre recombinase is efficiently activated by a one hour heat-shock in both the HSPCre1 and HSPCre13 strain. Therefore both strains can be used as activator strains in the binary Cre/loxP-system. A crossing of the C5 strain with the HSPCre1 strain revealed that transcripts of an inducible reporter gene could be detected already one hour after heat-shock-activation of the cre recombinase. Ubiquitous overexpression of the HNF1β derivative P328L329del by a one-hour heat-shock caused kidney defects and malformations of other organs in the mutant larvae, but this was not the case in all of these animals. This result was validated with additional crossings of independent activator and effector strains. The binary system was optimized by application of a two hour heat-shock and upon ubiquitous overexpression of P328L329del 100 % of the mutant larvae showed a kidney phenotype, which became manifested in defects particularly in the proximal tubules and in a significantly reduced kidney size. Additionally the overexpression of P328L329del interfered with the development of the gut, the tail and the eye. Furthermore the expression of the two proximal tubule markers slc3a1 and tmem27 was affected. Both genes play a crucial role in the renal absorption of amino acids. This finding reveals an unknown function of HNF1β in kidney development. The overexpression of the HNF1β derivative A263insGG also caused malformations of the kidney, gut, tail and eye in mutant larvae. In comparison to animals with activated P328L329del the kidney size was significantly increased. The overexpression of HNF1β had no effect on development. Furthermore two new effector strains for pronephros specific overexpression of HNF1β and the mutant derivative P328L329del were established. The pronephros specific overexpression of HNF1β led to kidney defects and gut malformations. In contrast to the ubiquitous overexpression no malformations of tail and eye were observed. In addition a new method was established where a local heat-shock is induced by laser beams. With this approach it was possible to activate the inducible EYFP reporter gene in single cells of the tail and in the pronephros. This method was applied in Xenopus laevis for the first time and allows for a tissue specific gene activation without the need of tissue specific promoters.
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ABSTRACT: Solute carriers (SLC) that transport amino acids are key players in health and diseases in humans. Their prokaryotic relatives are often involved in essential physiological processes in microorganisms, e.g. in homeostasis and acidic/osmotic stress response. High-resolution X-ray structures of the sequence-unrelated amino acid transporters unraveled a striking structural similarity between carriers, which were formerly assigned to different families. The highly conserved fold is characterized by two inverted structural repeats of five transmembrane helices each and indicates common mechanistic transport concepts if not an evolutionary link among a large number of amino acid transporters. Therefore, these transporters are classified now into the structural amino acid-polyamine-organocation superfamily (APCS). The APCS includes among others the mammalian SLC6 transporters and the heterodimeric SLC7/SLC3 transporters. However, it has to be noted that the APCS is not limited entirely to amino acid transporters but contains also transporters for, e.g. amino acid derivatives and sugars. For instance, the betaine-choline-carnitine transporter family of bacterial activity-regulated Na(+)- and H(+)-coupled symporters for glycine betaine and choline is also part of this second largest structural superfamily. The APCS fold provides different possibilities to transport the same amino acid. Arginine can be transported by an H(+)-coupled symport or by antiport mechanism in exchange against agmatine for example. The convergence of the mechanistic concept of transport under comparable physiological conditions allows speculating if structurally unexplored amino acid transporters, e.g. the members of the SLC36 and SLC38 family, belong to the APCS, too. In the kidney, which is an organ that depends critically on the regulated amino acid transport, these different SLC transporters have to work together to account for proper function. Here, we will summarize the basic concepts of Na(+)- and H(+)-coupled amino acid symport and amino acid-product antiport in the light of the respective physiological requirements.Current Topics in Membranes 01/2012; 70:1-28. DOI:10.1016/B978-0-12-394316-3.00001-6 · 1.77 Impact Factor