Comparative functional profiling of plant ACR3 orthologs reveals metalloid-sensitive trafficking and arsenic efflux capabilities

Arsenic contamination is a pervasive environmental and public health challenge. In plants from microalgae to gymnosperms, members of the ACR3 transporter family mediate arsenic tolerance, yet their evolution, diversity, and regulatory mechanisms remain largely unexplored. Here, ACR3 genes from phylogenetically distant Coccomyxa subellipsoidea (CsACR3), Raphidocelis subcapitata (RsACR3), Chlamydomonas eustigma (CeACR3), Physcomitrium patens (PpACR3), and Picea sitchensis (PsACR3) were cloned, heterologously expressed in budding yeast, and functionally characterized alongside previously reported ACR3 genes from Marchantia polymorpha (MpACR3) and Pteris vittata (PvACR3). Our in silico analysis confirms that the putative proteins share structural and sequence homology with characterized ACR3 transporters, with many family members harboring elongated N-terminal tails unique to plant orthologs. We confirm that the analyzed genes encode functional arsenic efflux transporters conferring distinct levels of arsenite and arsenate resistance in yeast cells. We show that, in yeast cells, CsACR3, RsACR3, and PvACR3 constitutively localize to the plasma membrane, whereas the subcellular localization of CeACR3, MpACR3, PpACR3, and PsACR3 is regulated by metalloid exposure, triggering their endoplasmic reticulum-to-plasma membrane trafficking. Using MpACR3 as a model, we prove that this transporter requires its N-terminal domain for the intracellular retention, which is dynamically alleviated specifically in response to metalloids. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=119 SRC="FIGDIR/small/694196v1_ufig1.gif" ALT="Figure 1"> View larger version (46K): org.highwire.dtl.DTLVardef@9b1435org.highwire.dtl.DTLVardef@14bb23forg.highwire.dtl.DTLVardef@a34addorg.highwire.dtl.DTLVardef@88186c_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIPlant ACR3 transporters act as arsenic efflux systems across diverse lineages. C_LIO_LIACR3 orthologs confer distinct arsenite and arsenate resistance in yeast. C_LIO_LIMetalloids trigger ER-to-PM trafficking of several plant ACR3 proteins. C_LIO_LIElongated N-termini regulate metalloid-responsive ACR3 localization. C_LIO_LIMpACR3 N-terminus functions as a metalloid-sensitive retention module. C_LI