Succinate-acetate permease from Citrobacter koseri is an anion channel that unidirectionally translocates acetate

Abstract

Acetate is an important metabolite in metabolism and cell signaling. Succinate-Acetate Permease (SatP) superfamily proteins are known to be responsible for acetate transport across membranes, but the nature of this transport remains unknown. Here, we show that the SatP homolog from Citrobacter koseri (SatP_Ck) is an anion channel that can unidirectionally translocate acetate at rates of the order of ~10 ions/s. Crystal structures of SatP_Ck in complex with multiple acetates at 1.8 Å reveal that the acetate pathway consists of four acetate-binding sites aligned in a single file that are interrupted by three hydrophobic constrictions. The bound acetates at the four sites are each orientated differently. The acetate at the cytoplasmic vestibule is partially dehydrated, whereas those in the main pore body are fully dehydrated. Aromatic residues within the substrate pathway may coordinate translocation of acetates via anion-π interactions. SatP_Ck reveals a new type of selective anion channel and provides a structural and functional template for understanding organic anion transport.

Characterization of the GPR1/FUN34/YaaH protein family in the green microalga Chlamydomonas suggests their role as intracellular membrane acetate channels

“…However, the N‐terminal regions (16–33 amino acids) of these five protein isoforms differ in length and amino acid composition (Figure a). The five protein sequences contain the typical signatures conserved in the GPR1/FUN34/YaaH superfamily (Pfam01184; PS01114; IPR000791) (Figure a, Table S1) (Qiu et al., ; Ribas et al., ). The secondary structure analysis indicates that Cr GFY1–5 proteins are integral, multipass, membrane proteins composed of six transmembrane segments (Figure a) with location of both N‐ and C‐terminal regions on the same side of the membrane, which is predicted to be cytosolic [Phobius (Käll, Krogh, & Sonnhammer, ); TOPCONS (Tsirigos, Peters, Shu, Käll, & Elofsson, ); I‐TASSER (Yang et al., )].…”

“…The predicted structures contain six alpha‐helical transmembrane (TM) segments forming an inner pore along with a soluble N‐terminal region. The servers suggest that the target‐template models share main structural features with the succinate‐acetate channel monomer of Citrobacter koseri (SatP_Ck, UniprotID: A8ALU5) (Qiu et al., ) and E. coli (SatP_Ec (also known as YaaH): P0AC98) (Sá‐Pessoa et al., ; Sun et al., ). These multipass membrane proteins also belong to GFY protein superfamily and their structures were recently resolved by X‐ray crystallography (PDB ID SatP_Ck: 5ys3; SatP_Ec: 5ZUG).…”

“…These small molecule protein channels were originally discovered in mutants of the yeast Yarrowia lipolytica , which were unable to grow on acetate. The involvement of GFY membrane proteins in acetate uptake has been demonstrated in Escherichia coli (enzyme: SatP_Ec or YaaH) (Sá‐Pessoa et al., ; Sun et al., ), Citrobacter koseri (SatP_Ck) (Qiu et al., ), Methanosarcina acetivorans (AceP) (Ribas et al., ), Saccharomyces cerevisiae (Ady2) (Paiva, Devaux, Barbosa, Jacq, & Casal, ), and Aspergillus nidulans (AcpA) (Robellet, Flipphi, Pégot, MacCabe, & Vélot, ). GFY proteins have also been implicated in acetate sensing for the yeast Y. lipolytica (Gpr1) (Augstein, Barth, Gentsch, Kohlwein, & Barth, ; Tzschoppe, Augstein, Bauer, Kohlwein, & Barth, ).…”

“…GFY proteins have also been implicated in acetate sensing for the yeast Y. lipolytica (Gpr1) (Augstein, Barth, Gentsch, Kohlwein, & Barth, ; Tzschoppe, Augstein, Bauer, Kohlwein, & Barth, ). Hallmarks of GFY proteins are six transmembrane domains and at least two distinct conserved amino acid signatures which have been determined to be directly involved in transport mechanisms by substrate interaction studies (Qiu et al., ; Ribas et al., ). Two recently resolved crystal structures of bacterial GFYs include the SatP_Ec from E. coli (Sá‐Pessoa et al., ; Sun et al., ) and SatP_Ck from Citrobacter koserii (Qiu et al., ).…”

“…Hallmarks of GFY proteins are six transmembrane domains and at least two distinct conserved amino acid signatures which have been determined to be directly involved in transport mechanisms by substrate interaction studies (Qiu et al., ; Ribas et al., ). Two recently resolved crystal structures of bacterial GFYs include the SatP_Ec from E. coli (Sá‐Pessoa et al., ; Sun et al., ) and SatP_Ck from Citrobacter koserii (Qiu et al., ). In C. reinhardtii , five GFY genes emerged as candidate acetate permeases in clusters of transcripts responsive to acetic acid (Goodenough et al., ).…”