HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Chillarón, J. Articles by Palacín, M. Search for Related Content PubMed PubMed Citation Articles by Chillarón, J. Articles by Palacín, M. Social Bookmarking                      What's this?

Volume 272, Number 14, Issue of April 4, 1997 pp. 9543-9549
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.

---

An Intracellular Trafficking Defect in Type I Cystinuria rBAT Mutants M467T and M467K

(Received for publication, May 29, 1996, and in revised form, January 22, 1997)

Josep Chillarón , Raúl Estévez , Ita Samarzija ^§ , Siegfried Waldegger ^§ , Xavier Testar , Florian Lang ^§ , Antonio Zorzano , Andreas Busch ^§ and Manuel Palacín

From the  Department of Biochemistry and Molecular Biology, Faculty of Biology, Universitat de Barcelona, Avda. Diagonal 645, Barcelona 08028, Spain and the ^§ Institute of Physiology, Eberhard-Karls-Universität Tübingen, Gmelinstrasse 5, D-72076 Tübingen, Germany

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 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.

CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?