Membrane topology of the cyanobacterial bicarbonate transporter, BicA, a member of the SulP (SLC26A) family

Figures & data

Table I.  Enzyme activity of BicA dual reporter fusions.

BicA phoA-lacZ fusion point^a	Colony colour^b	AP activity (% of max rate)^c	BG activity (% of max rate)^c	AP:BG ratio	Topology location score^d
G19	Red	0.4	50.6	0.01	cytoplasmic
A32	Blue	46.7	14.8	3.16	periplasmic
G34	Blue	nd	nd	–	periplasmic
A41	Blue	98.3	9.1	10.82	periplasmic
G43	Blue	100.0	34.7	2.89	periplasmic
W46	Blue	65.3	20.5	3.19	periplasmic
F53	Purple	38.0	77.8	0.49	TMH
G59	Red	0.9	24.3	0.04	cytoplasmic
V74	Purple	nd	nd	–	TMH
T84	Purple	4.8	24.5	0.20	TMH
G93	Blue	40.8	68.2	0.60	periplasmic
A97	Purple	29.7	75.6	0.39	TMH
V100	Blue	3.8	2.3	1.65	periplasmic
F107	Purple	0.4	33.5	0.01	cytoplasmic
T121	Red	0.5	38.6	0.01	cytoplasmic
V141	Purple	2.5	50.6	0.05	TMH
Q150	Blue	5.7	5.7	1.00	periplasmic
G154	Blue	31.2	34.7	0.90	periplasmic
N170	Blue	4.4	1.7	2.60	periplasmic
A177	Blue	nd	nd	–	periplasmic
P190	Purple	0.6	36.3	0.02	cytoplasmic
K192	Red	0.6	31.8	0.02	cytoplasmic
T208	Purple	9.4	69.3	0.14	TMH
I210	Purple	24.0	13.3	1.80	periplasmic
A211	Blue	48.0	13.6	3.52	periplasmic
D221	Blue	50.9	85.8	0.59	periplasmic
I225	Blue	14.8	33.3	0.44	periplasmic
V236	Blue	32.1	61.9	0.52	periplasmic
I249	Blue	22.3	83.5	0.27	TMH
V268	Red	0.2	63.2	0.00	cytoplasmic
E277	Red	0.6	100.0	0.01	cytoplasmic
K282	Red	nd	nd	–	cytoplasmic
G288	Red	0.2	13.4	0.01	cytoplasmic
G298	Red	nd	nd	–	cytoplasmic
V311	Red	1.6	17.2	0.09	cytoplasmic
N312	Red	0.2	16.4	0.01	cytoplasmic
G336	Purple	7.2	17.0	0.42	TMH
L340	Blue	44.7	4.1	10.90	periplasmic
L346	Purple	21.7	21.0	1.03	periplasmic
A347	Blue	nd	nd	–	periplasmic
I360	Red	19.3	44.8	0.43	TMH
A369	Red	0.9	68.5	0.01	cytoplasmic
A377	Purple	nd	nd	–	TMH
L385	Blue	nd	nd	–	periplasmic
V388	Blue	87.3	36.4	2.40	periplasmic
I393	Blue	72.0	44.8	1.61	periplasmic
Q415	Red-Purple	1.4	20.5	0.07	cytoplasmic
D426	Red-Purple	nd	nd	–	cytoplasmic
D440	Red-Purple	1.8	40.9	0.05	cytoplasmic
G458	Red-Purple	nd	nd	–	cytoplasmic
E466	Red-Purple	nd	nd	–	cytoplasmic
P561	Red-Purple	0.5	23.6	0.02	cytoplasmic

^aAmino acid position of the residue immediately before the phoA/lacZ reporter. ^bColour of colony as determined on dual indicator plates. ^cThe percentage of AP and BG activity relative to the maximum observed rates of AP or BG; activities were the mean of three independent cultures and SEs were less than ± 14% of the mean (n = 4 or greater). AP max rate = 81.2 nmoles MU/min/OD600 (G43 fusion); BG max rate = 7.76 nmoles MU/min/OD600 (E277 fusion); MU = 4-methylumbelliferone. ^dFusion locations were scored under the following criteria: Blue colonies with AP/BG ratios > 0.45 scores as periplasmic if AP activity was also > 5%; Purple colonies with AP/BG ratios between 0.1 and 2 were scored as TMH located; Red colonies with AP/BG ratios < 0.1 scored as cytoplasmic if AP activity was also < 2.5% of max.

Table II.  Predicted topologies for BicA using available prediction programs.

Prediction program	Number of TMH predicted	Location of N-terminus	Location of loop 8/9 region	Location of STAS domain
TMHMM	12	cytoplasmic	TMH	cytoplasmic
TopPred 2	12	cytoplasmic	TMH	cytoplasmic
OCTOPUS	10	external	TMH	external
SCAMPI-seq	11	cytoplasmic	TMH	external
SCAMPI-msa	12	cytoplasmic	cytoplasmic	cytoplasmic
MEMSAT3	12	cytoplasmic	cytoplasmic	cytoplasmic

Prediction server sites used are mentioned in the materials and methods section.

Figure 1.  The topology map for the Synechococcus PCC7002 Na⁺-dependent HCO₃^- transporter, BicA. Experimentally determined phoA/lacZ mapping positions (Table I) are shown as black (cytoplasmic), mid-grey (TMH located) or light grey (periplasmic); in the online version these are shown as blue, purple and red, respectively. The inset shows a WebLogo (weblogo.berkeley.edu) representation of the consensus of the ungapped alignment of the 50 residue loop between TMH 8 and 9; all the SulP/SLC26A members used to generate the phylogenetic tree shown in Figure 3 were used and BicA numbering is shown. This region corresponds to the second signature sequence identified by Saier et al. [23] for the SulP family; the most highly conserved subset, NSNKELIGQGLGN (279-291), is highlighted. This Figure is reproduced in colour in the online version of Molecular Membrane Biology.

Figure 2.  A simplified version of the topology map for BicA (Synechococcus PCC7002) showing the positions of positive charged residues and three highly conserved residues that are discussed in the text, E²⁸³ and N²⁹¹, that are implicated in human disease and T⁴⁸⁹, which is a putative phosphorylation site.

Figure 3.  Phylogenetic tree showing the relationship of the cyanobacterial BicA family (30 members) to the Arabidopsis SulP sulphate transporter family (13 members), the human SLC26A family (9 members) and a selection of bacterial BicA-like proteins (13 members). Protein sequences were aligned using ClustalW and nearest neighbour-joining routines in MEGA4 software (www.megasoftware.net). The scale marker represents 0.2 substitutions per residue.

Figure 4.  BicA (Synechococcus PCC7002) aligned to an extensively investigated member of the plant sulphate transporter family (SHST1, Stylosanthes hamata) and a member of the human SLC26A family (DTDST, SLC26A2) that is linked to the disease, diastrophic dysplasia. SCAMPI-msa predicted TMH regions are shaded grey based on separate queries for each protein. The two disease-causing residues, E²⁸³ and N²⁹¹, and the putative phosphorylation site T⁴⁸⁹, are shown.

Saier MH, Eng BH, Fard S, Garg J, Haggerty DA, Hutchinson WJ, Jack DL, Lai EC, Liu HJ, Nusinew DP, Omar AM, Pao SS, Paulsen IT, Quan JA, Sliwinski M, Tseng TT, Wachi S, Young GB. 1999. Phylogenetic characterization of novel transport protein families revealed by genome analyses. Biochim Biophys Acta – Rev Biomemb 1422:1–56.

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