Bioinformatic characterization of the 4-Toluene Sulfonate Uptake Permease (TSUP) family of transmembrane proteins

Abstract

The ubiquitous sequence diverse 4-Toluene Sulfonate Uptake Permease (TSUP) family contains few characterized members and is believed to catalyze the transport of several sulfur-based compounds. Prokaryotic members of the TSUP family outnumber the eukaryotic members substantially, and in prokaryotes, but not eukaryotes, extensive lateral gene transfer occurred during family evolution. Despite unequal representation, homologues from the three taxonomic domains of life share well-conserved motifs. We show that the prototypical eight TMS topology arose from an intragenic duplication of a four transmembrane segment (TMS) unit. Possibly, a two TMS α-helical hairpin structure was the precursor of the 4 TMS repeat unit. Genome context analyses confirmed the proposal of a sulfur-based compound transport role for many TSUP homologues, but functional outliers appear to be prevalent as well. Preliminary results suggest that the TSUP family is a member of a large novel superfamily that includes rhodopsins, integral membrane chaperone proteins, transmembrane electron flow carriers and several transporter families. All of these proteins probably arose via the same pathway: 2→4→8 TMSs followed by loss of a TMS either at the N- or C-terminus, depending on the family, to give the more frequent 7 TMS topology.

Figure 1

Phylogenetic tree of the 189 TSUP family proteins included in this study. The tree was generated using the ClustalX multiple alignment and FigTree programs. Protein abbreviations and their descriptions are listed in Table 1 in a clockwise fashion starting from cluster 1. The positions of individual proteins within the phylogenetic tree are revealed in Fig. S1.

Figure 2

16S/18S rRNA phylogenetic tree of genuses represented in this study. The Cloacamonas, Symbiobacterium, Oenococcus, Endoriftia and Desulforudis genera were excluded due to unreliable sequence data.

Figure 3

Portion of average hydropathy, amphipathicity, and similarity (AveHAS) plots for the 189 TSUP family proteins included in this study. A magnification of the TMS-containing region is presented due to the large size of the plot which reveals 8 clear TMSs. However, as many as 12 poorly conserved peaks of hydrophobicity can be seen, suggesting that some homologues have additional TMSs.

Figure 4

Demonstration that 8 TMS TSUP family members arose by intragenic duplication of a primordial 4 TMS encoding genetic element. (A) GAP alignment of TMSs 1–4 of TSUP Tko1 (Thermococcus kodakarensis; gi# 57640914) with TMSs 5–8 of the same protein. Initial identification of repeat units was done using the IC program. The GAP program was run with default settings and 500 random shuffles. Residue identities are signified by vertical lines, while close and more distant similarities are signified by colons or periods, respectively. The numbers at both ends of each line signify the positions of the residues in the proteins. TMS positions were predicted using the TMHMM 2.0 program for this and subsequent comparisons. A comparison score of 26 S.D. was obtained. (B) GAP alignment of TMSs 1–4 of TSUP Mch1 (Microcoleus chthonoplastes; gi# 224407624) with TMSs 5–8 of Mch1. A comparison score of 17 S.D. was obtained.

Figure 5

(A–B) The two best conserved motifs found within the TSUP family as predicted by MEME. Corresponding statistical scores are presented on the y-axis. MAST predictions of motifs based on the MEME results are presented on the x-axis below each motif graphic.

Figure 6

(A) Rhodopsin superfamily homology established through the use of GSAT/GAP and the Superfamily Principle. Established Rhodopsin superfamily proteins from TCDB and their homologues were used to establish homology between all members of the ten families. GSAT/GAP scores, adjacent to the arrows, are expressed in terms of standard deviations (S.D.). (B) Homology between members of the TSUP and LCT families. GAP alignment of TMSs 1–3 of TSUP Axy3 (Achromobacter xylosoxidans; gi 311107599) with TMSs 4–6 of LCT Asu1 (Ascaris suum; gi 324511247). A comparison score of 11.2 S.D. was obtained with 43.8% similarity and 28.1% identity. (C) Homology between members of the TSUP and NiCoT families. GAP alignment of TMSs 1–3 of TSUP Bja1 (Bradyrhizobium japonicum; gi 27376265) with TMSs 4–6 of NiCoT Pla1 (Parvibaculum lavamentivorans; gi 154252649). A comparison score of 12.8 S.D. was obtained with 44.7% similarity and 36.4% identity.

Figure 6

(A) Rhodopsin superfamily homology established through the use of GSAT/GAP and the Superfamily Principle. Established Rhodopsin superfamily proteins from TCDB and their homologues were used to establish homology between all members of the ten families. GSAT/GAP scores, adjacent to the arrows, are expressed in terms of standard deviations (S.D.). (B) Homology between members of the TSUP and LCT families. GAP alignment of TMSs 1–3 of TSUP Axy3 (Achromobacter xylosoxidans; gi 311107599) with TMSs 4–6 of LCT Asu1 (Ascaris suum; gi 324511247). A comparison score of 11.2 S.D. was obtained with 43.8% similarity and 28.1% identity. (C) Homology between members of the TSUP and NiCoT families. GAP alignment of TMSs 1–3 of TSUP Bja1 (Bradyrhizobium japonicum; gi 27376265) with TMSs 4–6 of NiCoT Pla1 (Parvibaculum lavamentivorans; gi 154252649). A comparison score of 12.8 S.D. was obtained with 44.7% similarity and 36.4% identity.

Figure 6

(A) Rhodopsin superfamily homology established through the use of GSAT/GAP and the Superfamily Principle. Established Rhodopsin superfamily proteins from TCDB and their homologues were used to establish homology between all members of the ten families. GSAT/GAP scores, adjacent to the arrows, are expressed in terms of standard deviations (S.D.). (B) Homology between members of the TSUP and LCT families. GAP alignment of TMSs 1–3 of TSUP Axy3 (Achromobacter xylosoxidans; gi 311107599) with TMSs 4–6 of LCT Asu1 (Ascaris suum; gi 324511247). A comparison score of 11.2 S.D. was obtained with 43.8% similarity and 28.1% identity. (C) Homology between members of the TSUP and NiCoT families. GAP alignment of TMSs 1–3 of TSUP Bja1 (Bradyrhizobium japonicum; gi 27376265) with TMSs 4–6 of NiCoT Pla1 (Parvibaculum lavamentivorans; gi 154252649). A comparison score of 12.8 S.D. was obtained with 44.7% similarity and 36.4% identity.