The Role of the STAS Domain in the Function and Biogenesis of a Sulfate Transporter as Probed by Random Mutagenesis

Abstract

Sulfate transporters in plants represent a family of proteins containing transmembrane domains that constitute the catalytic part of the protein and a short linking region that joins this catalytic moiety with a C-terminal STAS domain. The STAS domain resembles an anti-sigma factor antagonist of Bacillus subtilis, which is one distinguishing feature of the SLC26 transporter family; this family includes transporters for sulfate and other anions such as iodide and carbonate. Recent work has demonstrated that this domain is critical for the activity of Arabidopsis thaliana sulfate transporters, and specific lesions in this domain, or the exchange of STAS domains between different sulfate transporters, can severely impair transport activity. In this work we generated a Saccharomyces cerevisiae expression library of the A. thaliana Sultr1;2 gene with random mutations in the linking region-STAS domain and identified STAS domain lesions that altered Sultr1;2 biogenesis and/or function. A number of mutations in the ␤-sheet that forms the core of the STAS domain prevented intracellular accumulation of Sultr1;2. In contrast, the linking region and one surface of the STAS domain containing N termini of the first and second ␣-helices have a number of amino acids critical for the function of the protein; mutations in these regions still allow protein accumulation in the plasmamembrane, but the protein is no longer capable of efficiently transporting sulfate into cells. These results suggest that the STAS domain is critical for both the activity and biosynthesis/stability of the transporter, and that STAS sub-domains correlate with these specific functions.Sulfate transporters, designated Sultr (Sultr for the gene) in plants, are part of the SLC26 family of anion transporters that also includes iodide, chloride, and carbonate transporters and the motor protein prestin, which resides in the outer hair cells of the cochlea (1). SLC26 family proteins are predicted to have ten to fourteen transmembrane domains (TMDs) 2 and a C-terminal extension, designated as the STAS domain (2), that protrudes into the cytosol of the cell (3, 4). The STAS domain is joined to the catalytic domain of transporters by a linking (L) region, varies in length, and is not highly conserved relative to the catalytic domain. Some characteristics that make SLC26 transporters distinct from each other are likely to reside within unique features of L-STAS domains.Interestingly, the STAS domain of SLC26 transporter proteins resembles the bacterial anti-sigma factor antagonist SpoIIAA of Bacillus (2). SpoIIAA is a transcription factor that associates with the anti-sigma factor SpoIIAB; this association causes release of the sigma factor from SpoIIAB, triggering sporulation-specific transcription (5). SpoIIAB is also a kinase that can phosphorylate and inactivate SpoIIAA. Recently, STAS domains have been shown to be part of polypeptides other than anion transporters (6), suggesting that STAS sequences may serve a general regulatory function.STAS domains of SLC...

Slc26a9 Is Inhibited by the R-region of the Cystic Fibrosis Transmembrane Conductance Regulator via the STAS Domain

“…First, mutations within the ␤-pleated sheet region tend to reduce intracellular accumulation of the protein, suggesting that the STAS domain may also have a role in biosynthesis or stability of the Slc26 protein. Second, mutations of the N-terminal end of the ␣-helical regions was associated with impaired function despite expression at the plasma membrane (64). A more recent study of disease-associated mutations in the STAS domain of SLC26A3 indicates a predominant effect on biosynthesis, affecting different steps in the folding and/or trafficking pathway of the protein (16).…”

“…Random mutagenesis of a plant SLC26 sulfate transporter revealed two general classes of mutant effects within the STAS domain (64). First, mutations within the ␤-pleated sheet region tend to reduce intracellular accumulation of the protein, suggesting that the STAS domain may also have a role in biosynthesis or stability of the Slc26 protein.…”

Increased sulfate uptake by E. coli overexpressing the SLC26-related SulP protein Rv1739c from Mycobacterium tuberculosis

“…7A) for subsequent tests of targeted sulfhydryl modification with methanethiosulfonate (MTS) reagents. Similar cysteine scanning mutagenesis has defined residues important for the function or folding of the phylogenetically related sulfate transporters Sultr1;2 of A. thaliana (Shibagaki et al 2004;Shibagaki et al 2006) and SHST1 of the tropical legume Stylosanthes hamata (Howitt 2005). The IPTG-induced increment in Rv1739c-associated sulfate uptake was preserved in the protein products of all mutations introduced into the Cys-less polypeptide (Fig.…”

“…However, unlike the STAS domain requirement for sulfate transport by A. thaliana Sultr1;2 (Shibagaki et al 2004), the STAS domain was dispensable for RV1739c-associated enhancement of sulfate uptake. Moreover, modification of selected conserved residues homologous to those found important for sulfate transport by the plant SulP polypeptides SHST1 (Khurana et al 2000;Shelden et al 2003) and Sultr1;2 polypeptides (Shibagaki et al 2006) also appeared unimportant for Rv1739c-associated sulfate uptake. Expression of the Rv1739c STAS domain in the absence of its transmembrane domain did not enhance sulfate uptake.…”

“…Overexpression screening of several bacterial SulP genes revealed inducible, stable accumulation of M. tuberculosis Rv1739c and E. coli ychM. In view of the importance of the SulP gene family in plant (Yoshimoto et al 2007) and yeast (Shibagaki et al 2006), and fish biology (Renfro et al 1999;Nakada et al 2005;Katoh et al 2006), and the significance of the SLC26 gene superfamily in mammalian biology (Mount et al 2004;Markovich et al 2007), and the proposed importance of sulfate in M. tuberculosis pathogenicity (Schelle et al 2006;Fan et al 2007), we investigated further the function of Rv1739c (Fig. 1A) as a candidate sulfate transporter of mycobacteria.…”