Characteristics of cystine counter-transport in normal and cystinotic lysosome-rich leucocyte granular fractions

Gahl, W. A.; Tietze, Frank; Bashan, Nava; Bernardini, Isa; Raiford, David S.; Schulman, J D

doi:10.1042/bj2160393

Cited by 111 publications

(59 citation statements)

References 13 publications

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“…The disease phenotype led to the prediction that cystinosin is a cystine transporter localized in the lysosomal membrane (8). The construction of a cystinosin-GFP fusion protein has now allowed us to confirm the predicted subcellular localization due to the colocalization of this fusion protein with an antibody directed against the lysosomal membrane protein, LAMP-2.…”

Section: Discussionmentioning

confidence: 92%

“…One such example is the autosomal recessive disorder cystinosis (MIM 21980), which is characterized by an accumulation of intra-lysosomal cystine (7). Various biochemical studies over the years show that cystine transport across the lysosomal membrane is carrier-mediated, that the carrier is located in the membrane itself, and that it is distinct from the plasma membrane cystine transporters (8), which in contrast do not exclusively transport cystine (9,10). Moreover, the lysosomal cystine transporter is stimulated by the acid pH of the lysosome, which is ATP-dependent (11).…”

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confidence: 99%

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The Targeting of Cystinosin to the Lysosomal Membrane Requires a Tyrosine-based Signal and a Novel Sorting Motif

Cherqui

Kalatzis

Trugnan

et al. 2001

Journal of Biological Chemistry

122

120

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Cystinosis is a lysosomal transport disorder characterized by an accumulation of intra-lysosomal cystine. Biochemical studies showed that the lysosomal cystine transporter was distinct from the plasma membrane cystine transporters and that it exclusively transported cystine. The gene underlying cystinosis, CTNS, encodes a predicted seven-transmembrane domain protein called cystinosin, which is highly glycosylated at the N-terminal end and carries a GY-XX-⌽ (where ⌽ is a hydrophobic residue) lysosomal-targeting motif in its carboxyl tail. We constructed cystinosin-green fluorescent protein fusion proteins to determine the subcellular localization of cystinosin in transfected cell lines and showed that cystinosin-green fluorescent protein colocalizes with lysosomal-associated membrane protein 2 (LAMP-2) to lysosomes. Deletion of the GY-XX-⌽ motif resulted in a partial redirection to the plasma membrane as well as sorting to lysosomes, demonstrating that this motif is only partially responsible for the lysosomal targeting of cystinosin and suggesting the existence of a second sorting signal. A complete relocalization of cystinosin to the plasma membrane was obtained after deletion of half of the third cytoplasmic loop (amino acids 280 -288) coupled with the deletion of the GY-DQ-L motif, demonstrating the presence of the second signal within this loop. Using site-directed mutagenesis studies we identified a novel conformational lysosomalsorting motif, the core of which was delineated to YF-PQA (amino acids 281-285).

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Section: Discussionmentioning

confidence: 92%

mentioning

confidence: 99%

The Targeting of Cystinosin to the Lysosomal Membrane Requires a Tyrosine-based Signal and a Novel Sorting Motif

Cherqui

Kalatzis

Trugnan

et al. 2001

Journal of Biological Chemistry

122

120

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“…It is caused by a failure of cystine efflux from lysosomes (2-4), which results in intralysosomal accumulation of disulfides of the amino acid cysteine (5,6). Cystine efflux is inhibited by a defect in lysosomal membrane cystine transport (which is carrier-mediated) (7,8).…”

mentioning

confidence: 99%

Immunolocalization of Cystinosin, the Protein Defective in Cystinosis

Haq¹,

Kalatzis²,

Gubler³

et al. 2002

Journal of the American Society of Nephrology

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ABSTRACT. Cystinosis is an autosomal recessive disorder associated with excessive lysosomal cystine accumulation secondary to defective lysosomal cystine efflux. CTNS, the gene mutated in cystinosis, codes for the lysosomal membrane protein cystinosin. Antisera were raised in rabbits to a carboxy-terminal oligopeptide sequence from cystinosin. Antisera were screened by Western blotting and immunocytochemical analyses of transfected COS-7 cells expressing either human wild-type cystinosin, a wild-type cystinosin-green fluorescent protein (GFP) fusion protein, or a fusion protein of GFP and mutant human cystinosin with a carboxy-terminal deletion. In Western blots, bands corresponding to cystinosin or cystinosin-GFP were observed in transfected cells but no signal was detected in cells expressing the carboxy-terminal mutant; preimmune sera yielded negative results in all three cases. In transfected cells expressing wild-type cystinosin, immunoreactivity appeared in subcellular vesicles. In cells expressing the wild-type cystinosin-GFP fusion protein, immunoreactivity colocalized with GFP fluorescence. Previous studies demonstrated that GFP fluorescence from this construct colocalized with immunostaining for a known lysosomal membrane protein, i.e., lysosome-associated membrane protein 2. In immunohistochemical analyses, cystinosin localized to tubule epithelia in three normal human kidneys, with a pattern similar to that of lysosome-associated membrane protein 2; cystinosin immunoreactivity was absent in kidneys from patients with a CTNS deletion. For the first time, antisera have been raised that localize cystinosin in cells in vitro and in vivo.

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“…As IHKE-1 cells express the NHE-3 mRNA (Figure 7), it seems highly likely that the observed decrease in Na ϩ /H ϩ activity by CDME is due to interference with this isoform. The Na ϩ /H ϩ -exchanger, which functions in parallel with a Cl Ϫ -HCO 3 Ϫ -exchanger, is also involved in the reabsorption of the filtered HCO 3 Ϫ in the proximal tubule. Thus, its inhibition also plays a major role in the severe HCO 3 Ϫ losses, leading to metabolic acidosis (proximal tubular acidosis type 2) seen in cystinotic patients.…”

Section: Discussionmentioning

confidence: 99%

“…Cystinosis is an autosomal recessive disease in which a defect in the transport of the amino acid cystine out of the lysosomes leads to its intralysosomal accumulation (1)(2)(3)(4) in multiple organs, including the kidney (5). The lysosomal cystine transporter has been identified and named cystinosin (6).…”

mentioning

confidence: 99%

Inhibition of Na+-Dependent Transporters in Cystine-Loaded Human Renal Cells

Çetinkaya¹,

Schlatter²,

Hirsch³

et al. 2002

Journal of the American Society of Nephrology

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Abstract. Cystinosis is the most common cause of the renal Fanconi syndrome in children, leading to severe electrolyte disturbances and growth failure. A defective lysosomal transporter, cystinosin, results in intralysosomal accumulation of cystine. Loading cells with cystine dimethyl ester (CDME) is the only available model for this disease. This model was used to present electrophysiologic studies on immortalized human kidney epithelial (IHKE-1) cells that had been derived from the proximal tubule with the slow whole-cell patch clamp technique. Basal membrane voltages (V m ) of IHKE-1 cells were Ϫ30.7 Ϯ 0.4 mV (n ϭ 151). CDME concentration-dependently altered V m with an initial depolarization (2.7 Ϯ 0.2 mV; n ϭ 76; 1 mM CDME) followed by a more pronounced hyperpolarization (Ϫ9.9 Ϯ 1.0 mV; n ϭ 49). Three Na ϩ -dependent transporters were examined. Alanine (1 mM) depolarized IHKE-1 cells by 17.6 Ϯ 0.7 mV (n ϭ 59), and phosphate (1.8 mM) depolarized by 9.7 Ϯ 1.1 mV (n ϭ 18).Acidification of IHKE-1 cells with propionate (20 mM) resulted in a depolarization of V m by 7.1 Ϯ 0.3 mV (n ϭ 21) followed by a repolarization by 2.9 Ϯ 0.3 mV/min (n ϭ 17), reflecting Na ϩ /H ϩ -exchanger activity. Acute addition of 1 mM CDME did not alter the alanine-and propionate-induced changes in V m , but it reduced the phosphate-induced depolarization by 37 Ϯ 9% (n ϭ 10). Incubation with 1 mM CDME reduced the activity of all three transporters. Depolarizations by alanine and phosphate and the repolarization after propionate were inhibited by 57 Ϯ 4% (n ϭ 30), 45 Ϯ 9% (n ϭ 9), and 78 Ϯ 15% (n ϭ 8), respectively. In conclusion, this study demonstrates that CDME acutely alters V m of IHKE-1 cells and that at least three Na ϩ -dependent transporters are inhibited, the Na ϩ -phosphate cotransporter most sensitively. This might suggest that phosphate depletion and dissipation of the Na ϩ -gradient are involved in the development of the Fanconi syndrome of cystinosis.

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Characteristics of cystine counter-transport in normal and cystinotic lysosome-rich leucocyte granular fractions

Cited by 111 publications

References 13 publications

The Targeting of Cystinosin to the Lysosomal Membrane Requires a Tyrosine-based Signal and a Novel Sorting Motif

The Targeting of Cystinosin to the Lysosomal Membrane Requires a Tyrosine-based Signal and a Novel Sorting Motif

Immunolocalization of Cystinosin, the Protein Defective in Cystinosis

Inhibition of Na+-Dependent Transporters in Cystine-Loaded Human Renal Cells

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