Agranoff D , Monahan IM , Mangan JA , Butcher PD , Krishna S .

Wellcome Trust

	Figure 1. Putative distribution of membrane-spanning segments of Mramp to illustrate alignment of conserved residues with homologues. This sketch has been derived from detailed hydropathy analyses performed by Cellier et al. (reference 31). The precise number of membrane-spanning segments and topology of the COOH-terminal region are still uncertain. Mramp sequence was analyzed using the Kyte-Doolittle algorithm (window size of 16 amino acids). The highly conserved distribution of thermodynamically unfavored charged residues within transmembrane segments (M), which is a feature of eukaryotic Nramp homologues (reference 31), is also discernible in Mramp and other prokaryotic homologues (boxed residues). The positions of two adjacent glycine residues in M4 (region c, marked *) are known to be functionally important; in murine Nramp1, a G169D mutation is associated with the pathogen-susceptible phenotype (reference 3), while in Nramp2, a mutated neighboring glycine in the mk mouse (G185R; reference 36) and Belgrade (b) rat (reference 11) causes microcytic (iron deficiency) anemia, probably through steric and charge effects. Mramp contains a consensus transport motif (CTM) resembling the “EAA” box or “binding protein–dependent transport system's inner membrane component signature” (region f) (see reference 31). This motif [E,Q] [S,T,A]×2, 3X,G, [L,I,V,M,F,Y,A], 4X, [F,L,I,V], [P,K] is found in numerous bacterial periplasmic permeases and some eukaryotic multisubunit transporters (reference 31). Mutational analysis of three residues in this region (marked * in region f) in murine Nramp2 has demonstrated the functional importance of the first two residues (reference 10). Other highly conserved residues are shown in bold. DCT1 sequence = rat Nramp2; S. cerevisiae sequence = smf1. Sequence data are available from EMBL/GenBank/DDBJ under accession nos. U15184 (M. leprae), Z99106 (B. subtilis), U00096 (E. coli), U15929 (Smf1), AF008439 (DCT1), and L32185 (Nramp1, human).
	Figure 2. 65Zn2+ uptake by oocytes expressing Mramp. (A) Induction of 65Zn2+ uptake by Mramp in Xenopus oocytes injected with RNA (5 ng in 50 nl) transcribed from pXmramp. The difference in 65Zn2+ uptake between experimental and water (50 nl)–injected control oocytes is highly significant (P < 0.001). Inset shows a separate experiment in which 65Zn2+ uptake in Mramp cRNA–injected oocytes was compared with antisense cRNA (pmarm)-injected control oocytes (P = 0.025). (B) 65Zn2+ uptake in water-injected and THT1 (5 ng)–injected control oocytes. THT1 is the T. brucei hexose transporter. There was no significant difference in 65Zn2+ uptake between water- and THT1-injected groups (P = 0.5). (C) 2-DOG uptake in Mramp- (5 ng), THT1- (5 ng), and water-injected oocytes. THT1 induces significantly greater 2-DOG uptake than either Mramp (P < 0.002) or water (P < 0.002). There was no significant difference in uptakes between the water- and Mramp-injected oocytes. Displayed are mean values (± SE) of uptakes (10 oocytes per experimental condition).
	Figure 3. pH dependence of 65Zn2+ uptake induced by Mramp. 65Zn2+ uptake was assayed in Mramp-injected and control oocytes in media varying in pH (see Materials and Methods). The increase in 65Zn2+ uptake (Fold) is shown for Mramp-expressing oocytes compared with controls. Uptakes at pH 5.5 (P < 0.001) and pH 6.0 (P ≤ 0.009) were significantly greater than uptakes under remaining conditions. There were no significant differences in uptake at pH 5.0, 6.5, and 7.0 (P > 0.1). Displayed are mean values (± SE) of uptakes (10 oocytes per experimental condition).
	Figure 4. Substrate specificity for Mramp. (A) Abolition of 65Zn2+ uptake by Mn2+ in Mramp RNA–injected oocytes. Increase in uptake compared with control oocytes in the absence and presence of Mn2+ (10 mM as MnCl2; P = 0.01). (B) Uptake of 55Fe2+ by Mramp RNA–injected oocytes compared with water-injected controls (P < 0.001). (C) Influence of divalent cation competitors on 55Fe2+ (100 μM) uptake by Mramp RNA–injected oocytes. Data from one experiment. Displayed are mean values (± SE) of uptakes (10 oocytes per experimental condition).
	Figure 5. Growth of M. tuberculosis H37Rv in different cationic conditions. (A) RT-PCR for Mramp and y39 carried out on total RNA isolated from M. tuberculosis H37Rv cultured in media containing different Fe2+ and Cu2+ concentrations. Equal amounts of RNA template (30 ng) were used in each reaction (3 ng for 16S RNA experiments). Lane 1, low Fe2+(<1 μM); lane 2, medium Fe2+ (4 μM); lane 3, high Fe2+ (48 μM); lane 4, low Cu2+ (<0.5 μM); lane 5, medium Cu2+ (5 μM); lane 6, high Cu2+ (70 μM). (B) Bacterial growth after 5 wk culture at each metal ion concentration. (C) Top panel, RT-PCR for Mramp on total RNA isolated from axenically cultured and intracellular BCG. Equal amounts of RNA template (135 ng) were used in each reaction. Lane 1, template from extracellular (axenically cultured) BCG; lane 2, template from extracellular BCG pretreated with RNAse A; lane 3, template from intracellular BCG; lane 4, template from intracellular BCG pretreated with RNAse A. Bottom panel, RT-PCR for rRNA (16S) using amounts of total RNA template identical to top panel. Lane 1, template from extracellular (axenically cultured) BCG; lane 2, template from extracellular BCG pretreated with RNAse A; lane 3, template from intracellular BCG; lane 4, template from intracellular BCG pretreated with RNAse A.

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