Identification of critical residues for transport activity of Acr3p, the Saccharomyces cerevisiae As(III)/H⁺ antiporter

Abstract

Summary Acr3p is an As(III)/H+ antiporter from Saccharomyces cerevisiae belonging to the bile/arsenite/riboflavin transporter superfamily. We have previously found that Cys151 located in the middle of the fourth transmembrane segment (TM4) is critical for antiport activity, suggesting that As(III) might interact with a thiol group during the translocation process. In order to identify functionally important residues involved in As(III)/H + exchange, we performed a systematic alanine-replacement analysis of charged/polar and aromatic residues that are conserved in the Acr3 family and located in putative transmembrane segments. Nine residues (Asn117, Trp130, Arg150, Trp158, Asn176, Arg230, Tyr290, Phe345, Asn351) were found to be critical for proper folding and trafficking of Acr3p to the plasma membrane. In addition, we found that replacement of highly conserved Phe266 (TM7), Phe352 (TM9), Glu353 (TM9) and Glu380 (TM10) with Ala abolished transport activity of Acr3p, while mutation of Ser349 (TM9) to Ala significantly reduced the As(III)/H + exchange, suggesting an important role of these residues in the transport mechanism. Detailed mutational analysis of Glu353 and Glu380 revealed that the negatively charged residues located in the middle of transmembrane segments TM9 and TM10 are crucial for antiport activity. We also discuss a hypothetical model of the Acr3p transport mechanism.

Investigation of metal toxicity leads to the identification of novel translation associated genes in yeast (Saccharomyces cerevisiae)

“…The expression of URE2 at the protein levels is one of the main focuses of my thesis. Other genes that influence heavy metal and metalloid toxicity in S. cerevisiae includes ARsenicals Resistance 3 (ARR3), Thiol-Specific Antioxidant (TSA1) and Bile Pigment Transporter (BPT1) (Chae et al, 1993;Petrovic et al, 2000;Meng et al, 2004;Markowska et al, 2015;Irokawa et al, 2016). Toxic metals may trigger very specific pathways in living organisms.…”

“…For example, As resistance machineries in majority of microorganisms is mediated by four different types of transmembrane channels or transporters (Ghosh et al, 1999;Meng et al, 2004). In Escherichia coli arsenite efflux is controlled by ArsB (Meng et al, 2004), in Sinorhizobium meliloti arsenite is exported to the outer membrane via the Major Intrinsic Protein AqpSp (Yang et al, 2005) and in yeast, S. cerevisiae detoxification of arsenite is achieved by arsenite efflux either through Arr3p or through compartmentalization of glutathione-arsenic adducts into the vacuole by Yeast Cadmium Factor Protein (Ycf1p) or excretes into the outer membrane via glutathione S-transferases (GSTs) proteins such as Ure2p (Ghosh et al, 1999;Bousset et al, 2001;Markowska et al, 2015).…”