Identification of conformationally sensitive amino acids in the Na(+)/dicarboxylate symporter (SdcS).
The Na(+)/dicarboxylate symporter (SdcS) from Staphylococcus aureus is a homologue of the mammalian Na(+)/dicarboxylate cotransporters (NaDC1) from the solute carrier 13 (SLC13) family. This study examined succinate transport by SdcS heterologously expressed in Escherichia coli, using right-side-out (RSO) and inside-out (ISO) membrane vesicles. The K(m) values for succinate in RSO and ISO vesicles were similar, approximately 30 microM. The single cysteine of SdcS was replaced to produce the cysteine-less transporter, C457S, which demonstrated functional characteristics similar to those of the wild type. Single-cysteine mutants were made in SdcS-C457S at positions that are functionally important in mammalian NaDC1. Mutant N108C of SdcS was sensitive to chemical labeling by MTSET {[2-(trimethylammonium)ethyl]methanethiosulfonate} from both the cytoplasmic and extracellular sides, depending on the conformational state of the transporter, suggesting that Asn-108 may be found in the translocation pore of the protein. Mutant D329C was sensitive to MTSET in the presence of Na(+) but only from the extracellular side. Finally, mutant L436C was insensitive to MTSET, although changes in its kinetic properties indicate that this residue may be important in substrate binding. In conclusion, this work identifies Asn-108 as a key residue in the translocation pathway of the protein, accessible in different states from both sides of the membrane. Functional characterization of SdcS should provide useful structural as well as functional details about mammalian transporters from the SLC13 family.
Joshi
Aditya D
AD
Department of Molecular Physiology and Biophysics, Baylor College of Medicine, Houston, Texas 77030, USA.
Pajor
Ana M
AM
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000187:drug effects; D000596:Amino Acids / Q000378:metabolism*; D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry*; D001692:Biological Transport / Q000187:drug effects; D015153:Blotting, Western; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D003545:Cysteine / Q000235:genetics; D007700:Kinetics; D008698:Mesylates / Q000494:pharmacology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis / Q000187:drug effects; D050505:Mutant Proteins / Q000737:chemistry; D011487:Protein Conformation / Q000187:drug effects; D011817:Rabbits; D016415:Sequence Alignment; D012964:Sodium / Q000494:pharmacology; D013211:Staphylococcus aureus / Q000737:chemistry*; D013386:Succinates / Q000378:metabolism; D027981:Symporters / Q000737:chemistry* / Q000378:metabolism; D013997:Time Factors; D022161:Transport Vesicles / Q000187:drug effects / Q000378:metabolism
D000596:Amino Acids; D001426:Bacterial Proteins; D008698:Mesylates; D050505:Mutant Proteins; D013386:Succinates; D027981:Symporters; C109154:(2-(trimethylammonium)ethyl)methanethiosulfonate; D012964:Sodium; D003545:Cysteine