Abstract
The role of cysteine residues in the Na(+)/dicarboxylate co-transporter (NaDC-1) was tested using site-directed mutagenesis. The transport activity of NaDC-1 was not affected by mutagenesis of any of the 11 cysteine residues, indicating that no individual cysteine residue is necessary for function. NaDC-1 is sensitive to inhibition by the impermeant cysteine-specific reagent, p-chloromercuribenzenesulphonate (pCMBS). The pCMBS-sensitive residues in NaDC-1 are Cys-227, found in transmembrane domain 5, and Cys-476, located in transmembrane domain 9. Although cysteine residues are not required for function in NaDC-1, their presence appears to be important for protein stability or trafficking to the plasma membrane. There was a direct relationship between the number of cysteine residues, regardless of location, and the transport activity and expression of NaDC-1. The results indicate that mutagenesis of multiple cysteine residues in NaDC-1 may alter the shape or configuration of the protein, leading to alterations in protein trafficking or stability.
Full Text
The Full Text of this article is available as a PDF (145.9 KB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Bindslev N., Wright E. M. Histidyl residues at the active site of the Na/succinate co-transporter in rabbit renal brush borders. J Membr Biol. 1984;81(2):159–170. doi: 10.1007/BF01868980. [DOI] [PubMed] [Google Scholar]
- Chen J. G., Liu-Chen S., Rudnick G. External cysteine residues in the serotonin transporter. Biochemistry. 1997 Feb 11;36(6):1479–1486. doi: 10.1021/bi962256g. [DOI] [PubMed] [Google Scholar]
- Griffith D. A., Pajor A. M. Acidic residues involved in cation and substrate interactions in the Na+/dicarboxylate cotransporter, NaDC-1. Biochemistry. 1999 Jun 8;38(23):7524–7531. doi: 10.1021/bi990076b. [DOI] [PubMed] [Google Scholar]
- Kunkel T. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci U S A. 1985 Jan;82(2):488–492. doi: 10.1073/pnas.82.2.488. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Pajor A. M. Sequence and functional characterization of a renal sodium/dicarboxylate cotransporter. J Biol Chem. 1995 Mar 17;270(11):5779–5785. doi: 10.1074/jbc.270.11.5779. [DOI] [PubMed] [Google Scholar]
- Pajor A. M. Sodium-coupled transporters for Krebs cycle intermediates. Annu Rev Physiol. 1999;61:663–682. doi: 10.1146/annurev.physiol.61.1.663. [DOI] [PubMed] [Google Scholar]
- Pajor A. M., Sun N., Bai L., Markovich D., Sule P. The substrate recognition domain in the Na+/dicarboxylate and Na+/sulfate cotransporters is located in the carboxy-terminal portion of the protein. Biochim Biophys Acta. 1998 Mar 6;1370(1):98–106. doi: 10.1016/s0005-2736(97)00249-6. [DOI] [PubMed] [Google Scholar]
- Pajor A. M., Sun N. Characterization of the rabbit renal Na(+)-dicarboxylate cotransporter using antifusion protein antibodies. Am J Physiol. 1996 Dec;271(6 Pt 1):C1808–C1816. doi: 10.1152/ajpcell.1996.271.6.C1808. [DOI] [PubMed] [Google Scholar]
- Pajor A. M., Sun N., Valmonte H. G. Mutational analysis of histidine residues in the rabbit Na+/dicarboxylate co-transporter NaDC-1. Biochem J. 1998 Apr 1;331(Pt 1):257–264. doi: 10.1042/bj3310257. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Sur C., Schloss P., Betz H. The rat serotonin transporter: identification of cysteine residues important for substrate transport. Biochem Biophys Res Commun. 1997 Dec 8;241(1):68–72. doi: 10.1006/bbrc.1997.7771. [DOI] [PubMed] [Google Scholar]
- Yan R. T., Maloney P. C. Identification of a residue in the translocation pathway of a membrane carrier. Cell. 1993 Oct 8;75(1):37–44. [PubMed] [Google Scholar]
- Yan R. T., Maloney P. C. Residues in the pathway through a membrane transporter. Proc Natl Acad Sci U S A. 1995 Jun 20;92(13):5973–5976. doi: 10.1073/pnas.92.13.5973. [DOI] [PMC free article] [PubMed] [Google Scholar]