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

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 ~107 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.

Mass spectrometry sequencing of transfer ribonucleic acids by the comparative analysis of RNA digests (CARD) approach.

The comparative analysis of ribonucleic acid digests (CARD) approach for sequencing of transfer ribonucleic acids (tRNAs) is described. This method is enabled by the differential labeling of two tRNA populations. A set of reference tRNAs, whose complete sequences including modifications are known, are labeled with (16)O during enzymatic digestion. The second (candidate) set of tRNAs, whose sequence information is desired, is labeled with (18)O. By combining the two digests, digestion products that share the same sequence between the reference and candidate will appear as doublets separated by 2 Da. Sequence or modification differences between the two will generate singlets that can be further characterized to identify how the candidate sequence differs from the reference. Using CARD, ca. 80% of the tRNAs from the bacterium Citrobacter koseri can be sequenced using ribonuclease T1 with Escherichia coli tRNAs as the reference. During these studies, we also discovered a sequence error for Escherichia coli tRNA-Thr1, and use this method to confirm the correct sequence for that tRNA.

Surprises from an unusual CLC homolog.

The chloride channel (CLC) family is distinctive in that some members are Cl(-) ion channels and others are Cl(-)/H(+) antiporters. The molecular mechanism that couples H(+) and Cl(-) transport in the antiporters remains unknown. Our characterization of a novel bacterial homolog from Citrobacter koseri, CLC-ck2, has yielded surprising discoveries about the requirements for both Cl(-) and H(+) transport in CLC proteins. First, even though CLC-ck2 lacks conserved amino acids near the Cl(-)-binding sites that are part of the CLC selectivity signature sequence, this protein catalyzes Cl(-) transport, albeit slowly. Ion selectivity in CLC-ck2 is similar to that in CLC-ec1, except that SO(4)(2-) strongly competes with Cl(-) uptake through CLC-ck2 but has no effect on CLC-ec1. Second, and even more surprisingly, CLC-ck2 is a Cl(-)/H(+) antiporter, even though it contains an isoleucine at the Glu(in) position that was previously thought to be a critical part of the H(+) pathway. CLC-ck2 is the first known antiporter that contains a nonpolar residue at this position. Introduction of a glutamate at the Glu(in) site in CLC-ck2 does not increase H(+) flux. Like other CLC antiporters, mutation of the external glutamate gate (Glu(ex)) in CLC-ck2 prevents H(+) flux. Hence, Glu(ex), but not Glu(in), is critical for H(+) permeation in CLC proteins.