An intracellular trafficking defect in type-I cystinuria rBAT mutants Met467Thr and Met467Lys
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., a...
| Authors: | ; ; ; ; ; ; ; ; |
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| Format: | Article |
| Status: | Published version |
| Publication date: | 1997 |
| Country: | España |
| Institution: | Varias* (Consorci de Biblioteques Universitáries de Catalunya, Centre de Serveis Científics i Acadèmics de Catalunya) |
| Repository: | Recercat. Dipósit de la Recerca de Catalunya |
| OAI Identifier: | oai:recercat.cat:2445/177125 |
| Online Access: | https://hdl.handle.net/2445/177125 |
| Access Level: | Open access |
| Keyword: | Aminoàcids Proteïnes portadores Glicoproteïnes Metabolisme Amino acids Carrier proteins Glycoproteins Metabolism |
| _version_ | 1878741303856988160 |
|---|---|
| author | Chillarón Chaves, José Julio |
| author2 | Estévez Povedano, Raúl Samarzija, Ita Waldegger, Siegfried Testar, Xavier Lang, Florian Zorzano Olarte, Antonio Busch, Andreas Palacín Prieto, Manuel |
| author2_role | author author author author author author author author |
| author_browse | Busch, Andreas Chillarón Chaves, José Julio Estévez Povedano, Raúl Lang, Florian Palacín Prieto, Manuel Samarzija, Ita Testar, Xavier Waldegger, Siegfried Zorzano Olarte, Antonio |
| author_facet | Chillarón Chaves, José Julio Estévez Povedano, Raúl Samarzija, Ita Waldegger, Siegfried Testar, Xavier Lang, Florian Zorzano Olarte, Antonio Busch, Andreas Palacín Prieto, Manuel |
| author_role | author |
| collection | Recercat. Dipósit de la Recerca de Catalunya |
| description | 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. |
| eu_rights_str_mv | openAccess |
| format | article |
| id | ES_e2cdc2b20a1f727d04aba0845c262e25 |
| institution | Varias* (Consorci de Biblioteques Universitáries de Catalunya, Centre de Serveis Científics i Acadèmics de Catalunya) |
| instname_str | Varias* (Consorci de Biblioteques Universitáries de Catalunya, Centre de Serveis Científics i Acadèmics de Catalunya) |
| network_acronym_str | ES |
| network_name_str | España |
| oai_identifier_str | oai:recercat.cat:2445/177125 |
| publishDate | 1997 |
| publishDateSort | 1997 |
| publisher | American Society for Biochemistry and Molecular Biology |
| publisherStr | American Society for Biochemistry and Molecular Biology |
| reponame_str | Recercat. Dipósit de la Recerca de Catalunya |
| spelling | An intracellular trafficking defect in type-I cystinuria rBAT mutants Met467Thr and Met467Lys Chillarón Chaves, José Julio Estévez Povedano, Raúl Samarzija, Ita Waldegger, Siegfried Testar, Xavier Lang, Florian Zorzano Olarte, Antonio Busch, Andreas Palacín Prieto, Manuel Aminoàcids Proteïnes portadores Glicoproteïnes Metabolisme Amino acids Carrier proteins Glycoproteins Metabolism 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. American Society for Biochemistry and Molecular Biology https://hdl.handle.net/2445/177125 |
| spellingShingle | An intracellular trafficking defect in type-I cystinuria rBAT mutants Met467Thr and Met467Lys Chillarón Chaves, José Julio Aminoàcids Proteïnes portadores Glicoproteïnes Metabolisme Amino acids Carrier proteins Glycoproteins Metabolism |
| status_str | publishedVersion |
| title | An intracellular trafficking defect in type-I cystinuria rBAT mutants Met467Thr and Met467Lys |
| title_full | An intracellular trafficking defect in type-I cystinuria rBAT mutants Met467Thr and Met467Lys |
| title_fullStr | An intracellular trafficking defect in type-I cystinuria rBAT mutants Met467Thr and Met467Lys |
| title_full_unstemmed | An intracellular trafficking defect in type-I cystinuria rBAT mutants Met467Thr and Met467Lys |
| title_short | An intracellular trafficking defect in type-I cystinuria rBAT mutants Met467Thr and Met467Lys |
| title_sort | An intracellular trafficking defect in type-I cystinuria rBAT mutants Met467Thr and Met467Lys |
| topic | Aminoàcids Proteïnes portadores Glicoproteïnes Metabolisme Amino acids Carrier proteins Glycoproteins Metabolism |
| topic_facet | Aminoàcids Proteïnes portadores Glicoproteïnes Metabolisme Amino acids Carrier proteins Glycoproteins Metabolism |
| url | https://hdl.handle.net/2445/177125 |