Naslov
An intracellular trafficking defect in type i cystinuria rBAT mutants M467t and M467K

Autori
Chillaron, J. ; Estevez, R. ; Samaržija, Ita ; Waldegger, S. ; Testar, X. ; Lang, F. ; Zorzano, A. ; Busch, A. ; Palacin, M .

Izvornik
The Journal of biological chemistry (0021-9258) 272 (1997), 14; 9543-9549

Vrsta, podvrsta i kategorija rada
Radovi u časopisima, članak, znanstveni

Ključne riječi
Amino-acid transporter. Transmembrane conductance regulator. Xenopus-oocytes. Cystic-fibrosis. Rat-kidney. Expression cloning. Genetic-heterogeneity. Endoplasmic-reticulum. Glucose-transporter. Beta-subunit

Sažetak
The human rBAT protein elicits sodium-independent, high affinity obligatory exchange of cystine, dibasic amino acids, and some neutral amino acids in Xenopus oocytes (Chillaron, J., Estevez, R., Mora, C., Wagner, C. A., Suessbrich, H., Lang, F., Gelpi, J. L., Testar, X., Busch, A. E., Zorzano, A., and Palacin, M. (1996) J. Biol. Chem. 271, (17761-17770). Mutations in rBAT have been found to cause cystinuria (Calonge, M. J., Gasparini, P., Chillaron, J., Chillon, M., Galluci, M., Rousaud, F., Zelante, L., Testar, X., Dallapiccola, B., Di Silverio, F., Barcelo, P., Estivill, X., Zorzano, A., Nunes, V., and Palacin, 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 K-m 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 K-m, but the V-max 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.

Izvorni jezik
Engleski

Znanstvena područja
Temeljne medicinske znanosti

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