Multiple cysteine residues are necessary for sorting and transport activity of the arsenite permease Acr3p from Saccharomyces cerevisiae

Abstract

The yeast transporter Acr3p is a low affinity As(III)/H(+) and Sb(III)/H(+) antiporter located in the plasma membrane. It has been shown for bacterial Acr3 proteins that just a single cysteine residue, which is located in the middle of the fourth transmembrane region and conserved in all members of the Acr3 family, is essential for As(III) transport activity. Here, we report a systematic mutational analysis of all nine cysteine residues present in the Saccharomyces cerevisiae Acr3p. We found that mutagenesis of highly conserved Cys151 resulted in a complete loss of metalloid transport function. In addition, lack of Cys90 and Cys169, which are conserved in eukaryotic members of Acr3 family, impaired Acr3p trafficking to the plasma membrane and greatly reduced As(III) efflux, respectively. Mutagenesis of five other cysteines in Acr3p resulted in moderate reduction of As(III) transport capacities and sorting perturbations. Our data suggest that interaction of As(III) with multiple thiol groups in the yeast Acr3p may facilitate As(III) translocation across the plasma membrane.

Genome comparison of two Exiguobacterium strains from high altitude andean lakes with different arsenic resistance: identification and 3D modeling of the Acr3 efflux pump

“…S17 to arsenic would be the reduction of arsenate to arsenite and subsequent extrusion through membrane pumps as ArsB and Acr3 (Figure 7). Acr3 presents the conserved residues required for functionality as observed by Aaltonen and Silow (2008), Fu et al (2009), andMaciaszczyk-Dziubinska et al (2014) and would confer high resistance to arsenite (As [III]). Also, other mechanisms that overcome oxidative stress caused by arsenic would also help in the resistance to the metalloid.…”

Progress toward Understanding Protein S-acylation: Prospective in Plants

“…S-acylation of telomere-binding protein Rif1 anchored it to the inner nuclear membrane, which influences its role in heterochromatin dynamics (Park et al, 2011). Mutagenesis of cysteine in different positions of Arsenite permease Acr3p can cause its completely or partially dysfunction as a low affinity As(III)/H + and Sb(III)/H + antiporter, and Cys90 which localizes in the cytosolic loop but in close proximity to transmembrane regions has the high possibility to be S-acylated (Maciaszczyk-Dziubinska et al, 2014). It was also reported that S-acylation is necessary for the export of chitin synthase Chs3 from ER (Lam et al, 2006).…”

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

“…Following our previous characterization of the yeast Acr3p (Maciaszczyk-Dziubinska et al, 2014), we aimed to identify additional residues that are engaged in the As(III)/H + exchange. We hypothesized that conserved hydrophilic residues located in putative transmembrane regions of the yeast Acr3p might be involved in the As(III)/H + antiport.…”

“…The Acr3 family of arsenite transporters represents the most common pathway conferring high-level resistance to toxic metalloids (Rosen and Tamás, 2010;Maciaszczyk-Dziubinska et al, 2012). Members of the Acr3 family are present in bacteria, archaea, unicellular eukaryotes, fungi and lower plants but have been lost in flowering plants and animals (Fu et al, 2009;Indriolo et al, 2010;Maciaszczyk-Dziubinska et al, 2014). However, yeast or fern Acr3 can be successfully expressed in rice and Arabidopsis thaliana, leading to increased tolerance to arsenicals (Ali et al, 2012;Duan et al, 2012;Chen et al, 2013).…”

“…The Corynebacterium glutamicum Acr3 has also been shown to act as an As(III)/H + antiporter, strongly suggesting that the mechanism of As(III) transport is conserved among members of the Acr3 family (Villadangos et al, 2012). Moreover, a highly conserved cysteine residue (Cys129 in C. glutamicum, Cys138 in Alkaliphilus metalliredigens, Cys151 in S. cerevisiae) located in the middle of the fourth transmembrane segment (TM4) of Acr3p is crucial for transport activity (Fu et al, 2009;Villadangos et al, 2012;Maciaszczyk-Dziubinska et al, 2014). It has been hypothesized that a low-affinity interaction of As(III) with a single thiol group in Acr3 is necessary for the translocation process (Fu et al, 2009;Villadangos et al, 2012).…”