The conserved carboxy-terminal region of the ammonia channel AmtB plays a critical role in channel function

Figures & data

Figure 1.  Expression levels of AmtB in selected variants. (A) Western blots of membrane fractions from wild-type and a selection of AmtB variants. 5µg of protein was loaded in each lane. Estimated molecular weight of major band is indicated to the left of the panel. (B) Activity, in a washed assay, of each of the variants shown in panel A, expressed as a % of the wild-type.

Table I.  Relative [¹⁴C]-accumulation rates of GT1000 cells expressing AmtB CTR variants.

AmtB variant	Relative [^14C]-accumulation rate^a	Reactivity to αAmtB antiserum^b
AmtB/AmtB1	100%	+ + +
Δ(A406)	105%	+ + +
N405D	94%	+ +
AmtB4	31%	+ +
Δ(405–406)	12%	+ +
Δ(404–406)	21%	+ +
Δ(399–406)	43%	+ +
Δ(393–406)	31%	+ +
Δ(390–406)	28%	+ +
Δ(383–406)	27%	+ +
N405A	33%	+ +
N405Q	28%	+ +
N405A, Δ(A406)	18%	N.A.
Y404G	11%	+ +
G393D,Y404G	17%	N.A.
G393D,Δ(399–406)	38%	+ +
R384A	3%	+ + +
G393D	1%	+ + +
G393R	2%	+ + +
G393A	2%	+ + +
G393E	3%	+ + +
G393N	4%	+ + +
G393D, Δ(405–406)	6%	N.A.
ΔamtB	0%	N.D.

^a[¹⁴C]-accumulation rate determined using ‘washed’ assays. Figures are expressed as an average of 2–8 replicas (in which the replicas varied amongst themselves by less than 10%) and compared to data from a wild-type strain in the same assay. The mean activity of the wild-type strain, GT1000 (pSTUART1), was 0.225 nmol [¹⁴C]MA mg dry wt⁻¹ min⁻¹. ^bThe reactivity of variant proteins in cell membrane fractions to the αAmtB antiserum is expressed in relation to the band intensity for AmtB/AmtB1 and AmtB4. N.D. = not detected. N.A. = not analysed

Table II.  AmtB activities of representative variants measured in the ‘unwashed’ [¹⁴C]MA uptake assay.

AmtB variant	AmtB activity^a
	−MSF	+MSF
WT	100%	94%
N405Q	27%	31%
Δ(404–406)	21%	23%
Δ(393–406)	24%	20%
R384A	0%	3%
G393D	1%	0%

^aData expressed with reference to the wild-type strain, GT1000(pSTUART1), for which the mean activity was 0.1 nmol MA mg dry wt⁻¹ s⁻¹. All measurements are the average of 2–3 replicas in which the differences between replicate experiments were ≤15% of the mean. MSF (L-methionine sulphone) was added at a final concentration of 200µM.

Figure 2.  Detection of AmtBHA1, a HA-tagged derivative of AmtB, by western blotting. (A) Whole cell lysates (W), cytoplasmic (C) and membrane (M) fractions of GT1000(pGC2)-AmtB1, and GT1000(pESVHA1)-AmtBHA1, probed with αAmtB antiserum. (B) The same samples as in A, probed with αHA antibody. Estimated molecular weights of major bands are indicated to the left of the panels.

Figure 3.  Titration of the in vivo levels of AmtB variant proteins. Membrane fractions from GT1000(pESVHA1), GT1000(pESVHA2) and GT1000(pESVHA4) were freeze-thawed to disassemble AmtB into the monomer. Serial 2-fold dilutions of those preparations were probed with the αHA antibody. Amounts of protein loaded are indicated below the control in panel A. Estimated molecular weights of major bands are indicated to the left of the panels.

Figure 4.  Interaction of AmtB CTR variants with GlnK. Whole cell lysates (W), cytoplasmic (C) and membrane (M) fractions were probed with a αGlnK antiserum. -N: no NH₄Cl added; +N: after addition to the culture of 30 mM NH₄Cl for 15 min. The AmtB variants co-expressed with GlnK are indicated above the lanes. Data for representative variants are shown. Similar assays were carried out with Δ(399–406), R384A, G393D, G393R, Y404G, N405A and N405Q all of which showed no GlnK sequestration.

Figure 5.  Model for the CTR of E. coli AmtB. This model was derived as described in Materials and methods. (A) A single AmtB subunit showing the predicted CTR structure. The two helices of the CTR; from E387 to R391 and from D395 to S398 are labelled H1 and H2 respectively. (B) The cytoplasmic face of the AmtB trimer. Two monomers are shown as ribbons, the molecular surface of the third is shown in white and its cytoplasmic vestibule is indicated by the yellow circle (in on-line Figure). The CTR of each monomer is represented in red (in on-line Figure). (C) Hydrogen bonding (yellow dashed lines in on-line Figure) between Y404 and H196 (2.79 Å) and between G393, Q389 and N397. (D) Potential contacts between the H399, E401 and A403 side chains from one monomer (the core of the monomer has been removed) with the cytoplasmic face of the adjacent subunit of which the molecular surface is shown in yellow (in on-line Figure). This Figure is reproduced in colour in Molecular Membrane Biology on-line.

Supplementary Table I. Plasmid-encoded AmtB variants.

Plasmid	AmtB variant^a	CTR sequence^b	Reference
PSTUART1/2	AmtB	∼VGLRVPEEQEREGLDVNSHGENAYNA	This work
pGC2	AmtB1	∼VGLRVPEEQEREGLDVNSHGENAYNADL	(Coutts et al. 2002)
pGC4	AmtB4	∼SS	(Coutts et al. 2002)
pESV1	AmtB8	∼VGLRVPEEQEREGLDVNSHGENAYNADLH6	(Javelle et al. 2004)
PESVC1	Δ(A406)	∼VGLRVPEEQEREGLDVNSHGENAYN	This work
PESVC2	Δ(405–406)	∼VGLRVPEEQEREGLDVNSHGENAY	This work
PESVC3	Δ(404–406)	∼VGLRVPEEQEREGLDVNSHGENA	This work
PESVC4	Δ(399–406)	∼VGLRVPEEQEREGLDVNS	This work
PESVC5	Δ(393–406)	∼VGLRVPEEQERE	This work
PESVC6	Δ(390–406)	∼VGLRVPEEQ	This work
PESVC7	Δ(383–406)	∼VG	This work
PESVC8	N405A	∼VGLRVPEEQEREGLDVNSHGENAYAA	This work
PESVC9	N405A,Δ(A406)	∼VGLRVPEEQEREGLDVNSHGENAYA	This work
PESVC10	N405Q	∼VGLRVPEEQEREGLDVNSHGENAYQA	This work
PESVC11	N405D	∼VGLRVPEEQEREGLDVNSHGENAYDA	This work
PESVC13	Y404G	∼VGLRVPEEQEREGLDVNSHGENAGNA	This work
PESVC14	G393D	∼VGLRVPEEQEREDLDVNSHGENAYNA	This work
PESVC15	G393R	∼VGLRVPEEQERERLDVNSHGENAYNA	This work
PESVC16	G393A	∼VGLRVPEEQEREALDVNSHGENAYNA	This work
PESVC17	G393E	∼VGLRVPEEQEREELDVNSHGENAYNA	This work
PESVC18	G393N	∼VGLRVPEEQERENLDVNSHGENAYNA	This work
PESVC19	G393D,Δ(405–406)	∼VGLRVPEEQEREDLDVNSHGENAY	This work
PESVC20	G393D,Δ(399–406)	∼VGLRVPEEQEREDLDVNS	This work
PESVC22	G393D,Y404G	∼VGLRVPEEQEREDLDVNSHGENAGNA	This work
PESVC23	R384A	∼VGLAVPEEQEREGLDVNSHGENAYNA	This work
PESVHA1^c	HA AmtB	∼VGLRVPEEQEREGLDVNSHGENAYNA	This work
PESVHA2^c	HA Δ(405–406)	∼VGLRVPEEQEREGLDVNSHGENAY	This work
PESVHA4^c	HA N405Q	∼VGLRVPEEQEREGLDVNSHGENAYQA	This work

^aNumbering of the AmtB residues is based on the processed form of AmtB in which the signal peptide has been removed such that A23 of the preprotein becomes A1 of the mature protein. ^bV381to A406 in the wild-type protein. V381 is the first residue after the end of TMH11, as determined from the X-ray crystal structure of AmtB. Altered or added residues are shown in underlined bold type. ^cCarry the haemagglutinin (HA) epitope inserted between residues I86 and E87 in the first periplasmic loop of AmtB (see Supplementary Figure 1).

Supplementary Figure 1.  Topology of E. coli AmtB. Position of insertion of haemagglutinin (HA) epitope in some variants is indicated.

Supplementary Figure 2.  Sequence logo for the CTR of Amt proteins. The logo was generated using WebLogo (Crooks et al. 2004) from a CLUSTALW alignment of 51 Amt proteins including 27 from bacteria, 7 from archaea, 4 from fungi, 9 from plants, and 4 from C. elegans. L5 in the logo alignment is equivalent to L383 in E. coli AmtB and the highly conserved glycine residue, G15 in the logo, is G393.