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Background: Cystinuria is a heritable disorder of amino acid transport characterized by the defective transport of cystine and the dibasic amino acids through the brush border epithelial cells of the renal tubule and intestine tract. Three types of cystinuria (I, II, and III) have been described based on the urinary excretion of cystine and dibasic amino acids in obligate heterozygotes. The SLC3A1 gene coding for an amino acid transporter named rBAT is responsible for type I cystinuria, whereas the SLC7A9 gene coding for a subunit (b0,+AT) of rBAT is involved in determining non-type I (types II and III) cystinuria.Methods: The SLC3A1 gene sequence was investigated in a sample of seven type I/type I, three type I/non-type I, six type I/untyped, and four untyped unrelated cystinuric patients by RNA single-strand conformation polymorphism (RNA-SSCP).Results: Eight new point mutations (S168X, 765+1G>T, 766-2A>G, R452Q, Y461X, S547W, L564F, and C673W) and seven previously reported mutations were detected. These new mutations increase the number of mutated alleles so far characterized in SLC3A1 to 62.Conclusions: We have found SLC3A1 mutations in 0.739 of the type I chromosomes studied. The relatively high proportion of uncharacterized type I chromosomes suggests either that there may be mutations not yet found in SLC3A1 or that many of the assigned type I chromosomes in mixed type I/non-type I patients may have mutations in SLC7A9. If the hypothesis is excluded in the future, we believe that a third gene may be involved in cystinuria. [ABSTRACT FROM AUTHOR]

Calcineurin homologous protein 1 (CHP1) is a widely expressed, 22-kDa myristoylated EF-hand Ca(2+)-binding protein that shares a high degree of similarity with the regulatory B subunit of calcineurin (65%) and with calmodulin (59%). CHP1 localizes to the plasma membrane, the Golgi apparatus, and the nucleus and functions to regulate trafficking of early secretory vesicles, activation of T cells, and expression and transport of the Na-H exchanger NHE1. Although CHP1 contains nuclear export signals, whether its nuclear and cytoplasmic localization is regulated and has distinct functions remain unknown. We show that CHP1 is predominantly in the nucleus in quiescent fibroblasts, is translocated to cytoplasmic compartments with growth medium, and that translocation is inhibited by mutations in the nuclear export motifs. In a screen for proteins co-precipitating with CHP1 in quiescent cells we identified the upstream binding factor UBF, a DNA-binding protein and component of the RNA polymerase I complex regulating RNA synthesis. The CHP1-UBF interaction is restricted to the nucleus and inhibited by Ca(2+). Nuclear retention of CHP1 attenuates the abundance of UBF in the nucleolus and inhibits RNA synthesis when quiescent cells are transferred to growth medium. These data show UBF as a newly identified CHP1-binding protein and regulation of RNA synthesis as a newly identified function for nuclear-localized CHP1, which is distinct from CHP1 functions in the cytosol.

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-.

Heteromeric amino acid transporters (HATs) are composed of a heavy (SLC3 family) and a light (SLC7 family) subunit. Mutations in system b(0,+) (rBAT-b(0,+)AT) and in system y(+)L (4F2hc-y(+)LAT1) cause the primary inherited aminoacidurias (PIAs) cystinuria and lysinuric protein intolerance, respectively. Recent developments [including the identification of the first Hartnup disorder gene (B0AT1; SLC6A19)] and knockout mouse models have begun to reveal the basis of renal and intestinal reabsorption of amino acids in mammals.

Heteromeric amino acid transporters are composed of a heavy and a light subunit linked by a disulfide bridge. 4F2hc/xCT elicits sodium-independent exchange of anionic L-cysteine and L-glutamate (system x(c)(-)). Based on the accessibility of single cysteines to 3-(N-maleimidylpropionyl)biocytin, we propose a topological model for xCT of 12 transmembrane domains with the N and C termini located inside the cell. This location of N and C termini was confirmed by immunofluorescence. Studies of biotinylation and accessibility to sulfhydryl reagents revealed a re-entrant loop within intracellular loops 2 and 3. Residues His(110) and Thr(112), facing outside, are located at the apex of the re-entrant loop. Biotinylation of H110C was blocked by xCT substrates, by the nontransportable inhibitor (S)-4-carboxyphenylglycine, and by the impermeable reagent (2-sulfonatoethyl) methanethiosulfonate, which produced an inactivation of H110C that was protected by L-glutamate and L-cysteine with an IC(50) similar to the K(m). Protection was temperatureindependent. The data indicate that His(110) may lie close to the substrate binding/permeation pathway of xCT. The membrane topology of xCT could serve as a model for other light subunits of heteromeric amino acid transporters.

We measured sensitivity to thiol modification of the heteromeric glutamate/cystine transporter 4F2hc-xCT expressed in Xenopus oocytes. p-Chloromercuribenzoate (pCMB) and p-chloromercuribenzenesulfonate (pCMBS) rapidly blocked transport activity. Cys(327), located in the middle of the eighth transmembrane domain of the light subunit (xCT), was found to be the main target of inactivation. Cysteine, an impermeant reducing reagent, reversed pCMB and pCMBS effects only when applied from the extracellular medium. l-Glutamate and l-cystine, but not l-arginine, protected from the inactivation with an IC(50) similar to the K(m). Protection was not temperature-dependent, suggesting that it did not depend on large substrate-induced conformational changes. Mutation of Cys(327) to Ala and Ser slightly modified the K(m) and a C327L mutant abolished transport function without compromising transporter expression at the plasma membrane. The results indicate that Cys(327) is a functionally important residue accessible to the aqueous extracellular environment and is structurally linked to the permeation pathway and/or the substrate binding site.