Abstract
The human rBAT protein elicits sodium-independent, high affinity obligatory exchange of cystine, dibasic amino acids, and some neutral amino acids in Xenopus oocytes (Chillarón, J., Estévez, R., Mora, C., Wagner, C. A., Suessbrich, H., Lang, F., Gelpí, J. L., Testar, X., Busch, A. E., Zorzano, A., and Palacín, M. (1996) J. Biol. Chem. 271, 17761-17770). Mutations in rBAT have been found to cause cystinuria (Calonge, M. J., Gasparini, P., Chillarón, J., Chillón, M., Galluci, M., Rousaud, F., Zelante, L., Testar, X., Dallapiccola, B., Di Silverio, F., Barceló, P., Estivill, X., Zorzano, A., Nunes, V., and Palacín, M. (1994) Nat. Genet. 6, 420-426). We have performed functional studies with the most common point mutation, M467T, and its relative, M467K, using the oocyte system. The Km and the voltage dependence for transport of the different substrates were the same in both M467T and wild type-injected oocytes. However, the time course of transport was delayed in the M467T mutant: maximal activity was accomplished 3-4 days later than in the wild type. This delay was cRNA dose-dependent: at cRNA levels below 0.5 ng the M467T failed to achieve the wild type transport level. The M467K mutant displayed a normal Km, but the Vmax was between 5 and 35% of the wild type. The amount of rBAT protein was similar in normal and mutant-injected oocytes. In contrast to the wild type, the mutant proteins remained endoglycosidase H-sensitive, suggesting a longer residence time in the endoplasmic reticulum. We quantified the amount of rBAT protein in the plasma membrane by surface labeling with biotin 2 and 6 days after injection. Most of the M467T and M467K protein was located in an intracellular compartment. The converse situation was found in the wild type. Despite the low amount of M467T protein reaching the plasma membrane, the transport activity at 6 days was the same as in the wild type-injected oocytes. The increase in plasma membrane rBAT protein between 2 and 6 days was completely dissociated from the rise in transport activity. These data indicate impaired maturation and transport to the plasma membrane of the M467T and M467K mutant, and suggest that rBAT alone is unable to support the transport function.
Highlights
Brane of the M467T and M467K mutant, and suggest that rBAT alone is unable to support the transport function
An antisense sequence against rBAT abolishes in vivo the b0,ϩ-like activity expressed apically in these cells [8]. All these data indicate a role of rBAT in cystinuria. This common inheritable disorder is due to the defective transport of cystine and dibasic amino acids through the epithelial cells of the renal tubule and the intestinal tract (9 –11): the low solubility of cystine leads to the development of cystine calculi in the kidney
Attempts to determine transport over a longer period failed because of the increased oocyte mortality, so we cannot preclude the possibility that the M467K could reach the wild type uptake values
Summary
Uptake Experiments and Electrophysiological Studies—Oocyte management, injections, uptake measurements, and electrophysiological studies were as described elsewhere [1, 22]. The supernatant was dialyzed overnight at 4 °C against streptavidin buffer (SAv buffer) (0.3% Nonidet P-40, 500 mM NaCl, 1 mM CaCl2, 1 mM MgCl2, 10 mM Tris, pH 8, plus 2 M leupeptin and pepstatin and 1 mM phenylmethylsulfonyl fluoride) and centrifuged for 30 min at 14,000 rpm at 4 °C to remove insoluble material. SDS-PAGE was performed on total membrane proteins (endo Htreated or not) and the supernatants and pellets from the biotin-labeling experiments according to Laemmli [27]. For 1integrin Western blotting, nonreduced samples were transferred as above, blocked in 10% non-fat dry milk and 0.05% Tween-20 in phosphate-buffered saline for 30 min at 37 °C, and incubated with the monoclonal antibody 8C8 [29] at 1:10 dilution in the blocking solution overnight at room temperature. NHS-LC-biotin was from Pierce, and streptavidin-agarose beads were from Sigma
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