SLC26A9 is selected for endoplasmic reticulum associated degradation (ERAD) via Hsp70-dependent targeting of the soluble STAS domain

SLC26A9, a member of the solute carrier protein family, transports chloride ions across various epithelia. SLC26A9 also associates with other ion channels and transporters linked to human health, and in some cases these heterotypic interactions are essential to support the biogenesis of both proteins. Therefore, understanding how this complex membrane protein is initially folded might provide new therapeutic strategies to overcome deficits in the function of SLC26A9 partners, one of which is associated with Cystic Fibrosis. To this end, we developed a novel yeast expression system for SLC26A9. This facile system has been used extensively with other ion channels and transporters to screen for factors that oversee protein folding checkpoints. As commonly observed for other channels and transporters, we first noted that a substantial fraction of SLC26A9 is targeted for endoplasmic reticulum associated degradation (ERAD), which destroys folding-compromised proteins in the early secretory pathway. We next discovered that ERAD selection requires the Hsp70 chaperone, which can play a vital role in ERAD substrate selection. We then created SLC26A9 mutants and found that the transmembrane-rich domain of SLC26A9 was quite stable, whereas the soluble cytosolic STAS domain was responsible for Hsp70-dependent ERAD. To support data obtained in the yeast model, we were able to recapitulate Hsp70-facilitated ERAD of the STAS domain in human tissue culture cells. These results indicate that a critical barrier to nascent membrane protein folding can reside within a specific soluble domain, one that is monitored by components associated with the ERAD machinery.

Structure and function of an Arabidopsis thaliana sulfate transporter

Plant sulfate transporters (SULTR) mediate absorption and distribution of sulfate (SO42−) and are essential for plant growth; however, our understanding of their structures and functions remains inadequate. Here we present the structure of a SULTR from Arabidopsis thaliana, AtSULTR4;1, in complex with SO42− at an overall resolution of 2.8 Å. AtSULTR4;1 forms a homodimer and has a structural fold typical of the SLC26 family of anion transporters. The bound SO42− is coordinated by side-chain hydroxyls and backbone amides, and further stabilized electrostatically by the conserved Arg393 and two helix dipoles. Proton and SO42− are co-transported by AtSULTR4;1 and a proton gradient significantly enhances SO42− transport. Glu347, which is ~7 Å from the bound SO42−, is required for H+-driven transport. The cytosolic STAS domain interacts with transmembrane domains, and deletion of the STAS domain or mutations to the interface compromises dimer formation and reduces SO42− transport, suggesting a regulatory function of the STAS domain.

STAS Domain Only Proteins in Bacterial Gene Regulation

Sulfate Transport Anti-Sigma antagonist domains (Pfam01740) are found in all branches of life, from eubacteria to mammals, as a conserved fold encoded by highly divergent amino acid sequences. These domains are present as part of larger SLC26/SulP anion transporters, where the STAS domain is associated with transmembrane anchoring of the larger multidomain protein. Here, we focus on STAS Domain only Proteins (SDoPs) in eubacteria, initially described as part of the Bacillus subtilisRegulation of Sigma B (RSB) regulatory system. Since their description in B. subtilis, SDoPs have been described to be involved in the regulation of sigma factors, through partner-switching mechanisms in various bacteria such as: Mycobacterium. tuberculosis, Listeria. monocytogenes, Vibrio. fischeri, Bordetella bronchiseptica, among others. In addition to playing a canonical role in partner-switching with an anti-sigma factor to affect the availability of a sigma factor, several eubacterial SDoPs show additional regulatory roles compared to the original RSB system of B. subtilis. This is of great interest as these proteins are highly conserved, and often involved in altering gene expression in response to changes in environmental conditions. For many of the bacteria we will examine in this review, the ability to sense environmental changes and alter gene expression accordingly is critical for survival and colonization of susceptible hosts.

Structural basis for functional interactions in dimers of SLC26 transporters

The SLC26 family of transporters maintains anion equilibria in all kingdoms of life. The family shares a 7 + 7 transmembrane segments inverted repeat architecture with the SLC4 and SLC23 families, but holds a regulatory STAS domain in addition. While the only experimental SLC26 structure is monomeric, SLC26 proteins form structural and functional dimers in the lipid membrane. Here we resolve the structure of an SLC26 dimer embedded in a lipid membrane and characterize its functional relevance by combining PELDOR distance measurements and biochemical studies with MD simulations and spin-label ensemble refinement. Our structural model reveals a unique interface different from the SLC4 and SLC23 families. The functionally relevant STAS domain exerts a stabilizing effect on regions central in this dimer. Characterization of heterodimers indicates that protomers in the dimer functionally interact. The combined structural and functional data define the framework for a mechanistic understanding of functional cooperativity in SLC26 dimers.