Transmembrane topology of the arsenite permease Acr3 from Saccharomyces cerevisiae

Abstract

Acr3 is a plasma membrane transporter, a member of the bile/arsenite/riboflavin transporter (BART) superfamily, which confers high-level resistance to arsenicals in the yeast Saccharomyces cerevisiae. We have previously shown that the yeast Acr3 acts as a low affinity As(III)/H and Sb(III)/H antiporter. We have also identified several amino acid residues that are localized in putative transmembrane helices (TM) and appeared to be critical for the Acr3 activity. In the present study, the topology of Acr3 was investigated by insertion of glycosylation and factor Xa protease cleavage sites at predicted hydrophilic regions. The analysis of the glycosylation pattern and factor Xa cleavage products of resulting Acr3 fusion constructs provide evidence supporting a topological model of Acr3 with 10 TM segments and cytoplasmically oriented N- and C-terminal domains. Next, we investigated the role of the hydrophilic loop connecting TM8 and TM9, the large size of which is unique to members of the yeast Acr3 family of metalloid transporters. We found that a 28 amino acid deletion in this region does not affect Acr3 folding, trafficking substrate binding, or transport activity. Finally, we constructed a homology-based structural model of Acr3 using the crystal structure of the Yersinia frederiksenii homologue of the human bile acid sodium symporter ASBT.

Interactions with Arsenic: Mechanisms of Toxicity and Cellular Resistance in Eukaryotic Microorganisms

“…According to some authors [98,127,128], the As(III) efflux system based in the arsenite permease Acr3p is the major detoxification pathway in yeasts. Homologues of Acr3 are particularly widespread in archaea, bacteria, unicellular eukaryotes, fungi, and lower plants, but are absent in flowering plants and animals [129]. In most of bacteria and archaea, the transporter ArsB carries out this function.…”

“…Ycf1p could be a GSH conjugate transporter; it transports GSH-conjugated substrates across the vacuolar membrane, sequestering them within the vacuolar lumen [128]. flowering plants and animals [129]. In most of bacteria and archaea, the transporter ArsB carries out this function.…”

Transporters: the molecular drivers of arsenic stress tolerance in plants

“…ACR3 transporter is a plasma membrane transporter, belonging to the bile/arsenite/riboflavin transporter (BART) superfamily, with 10 transmembrane (TM) segments and cytoplasmically oriented N-and C-terminal domain (Wawrzycka et al 2017). It is present in bacteria, archaeabacteria, fungi and in some plants, where it acts as the main As(III) efflux pump (Maciaszczyk-Dziubinska et al 2014).…”

“…ACR3 exhibited As efflux capability to external environment, also reported by Daun et al (2012) in rice root. Cys151, Cys90 and Cys169 located in the fourth transmembrane span (TM4) and within the cytosolic loops of the transporter respectively are indispensable for full transport activity, while the hydrophilic loop connecting TM8 and TM9 is not involved in the transport activity (Wawrzycka et al 2017). Moreover, it has been suggested that the highly conserved residues Cys151 (TM4) and Ser349 (TM9) lie exactly within the intersection of the unwound fragments of the helices where they may form an As binding site.…”

Partial proteolysis improves the identification of the extracellular segments of transmembrane proteins by surface biotinylation

“…Amino acid mutagenesis gives structural information on a single position by modifying the particular residue using membrane-permeable and impermeable residue-specific chemical agents. Other approaches are N-glycosylation motif 25,26 (NXS/T where X can be any amino acid except proline) and epitope [27][28][29][30] insertion techniques, often employed for gathering topology data. N-glycosylation can only occur on the extra-cytosolic side of the membrane, and epitopes can be detected by specific antibodies, with or without permeabilization of the cell membrane.…”