Results: 5

1.
Fig. 2.

Fig. 2. From: Histidine pairing at the metal transport site of mammalian ZnT transporters controls Zn2+ over Cd2+ selectivity.

Zn2+ binding site. (A) Sequence alignment of YiiP and human ZnTs as indicated. The coordination residues of the transport site are highlighted in yellow for three invariant positions, and in green/purple for the fourth position that alternates between a histidine and aspartate, respectively. (B) Comparison between the metal binding sites of mammalian ZnTs (18) and bacterial YiiP (25). Histidine residues are colored red and aspartic acid residues are colored blue.

Eitan Hoch, et al. Proc Natl Acad Sci U S A. 2012 May 8;109(19):7202-7207.
2.
Fig. 4.

Fig. 4. From: Histidine pairing at the metal transport site of mammalian ZnT transporters controls Zn2+ over Cd2+ selectivity.

Functional conservation of the selectivity determinant in YiiP. (A) Normalized Fluozin-1 fluorescence in response to extravesicular Zn2+ exposure and EDTA addition as indicated. Fluorescence signals recorded from YiiP (red trace) or YiiPD45H (blue trace) proteoliposomes were normalized to their respective basal levels to remove the contribution of small differences in proteoliposome-loading. (B) Normalized Fluozin-1 fluorescence in response to extravesicular Cd2+ exposure and EDTA addition. (C) Zn2+ uptake rate of YiiP and YiiPD45H (mean ± s.e., n = 4), calculated using the fluorescence rising phase in Fig  3A. (D) Cd2+ uptake rate of YiiP and YiiPD45H (mean ± s.e., n = 4). Note that a single D-to-H mutation confers Zn2+ selectivity over Cd2+. (* relative to control; P ≤ 0.01, # relative to YiiP; P ≤ 0.01).

Eitan Hoch, et al. Proc Natl Acad Sci U S A. 2012 May 8;109(19):7202-7207.
3.
Fig. 1.

Fig. 1. From: Histidine pairing at the metal transport site of mammalian ZnT transporters controls Zn2+ over Cd2+ selectivity.

Zn2+ transport selectivity against Cd2+ (A) Normalized Fluozin-3 fluorescence changes in response to Zn2+ loading and withdrawal as indicated. The blue and red trace is recorded from HEK293-T cells transfected with wild-type ZnT5 and pcDNA control vector, respectively. The Inset shows a rapid ZnT5-mediated Zn2+ efflux from the cytoplasm upon Zn2+ withdrawal. (B) Normalized Fura-2 fluorescence changes in response to Cd2+ loading and withdrawal. TheInset shows no difference in Cd2+ effluxes recorded from the ZnT5-expressing cells (blue trace) and the control (red trace). (C) Rates of ZnT5-mediated Zn2+ and Cd2+ efflux normalized to the control rate as indicated (mean ± s.e., n = 3). (D) Zinpyr-1 fluorescence imaging of vesicular Zn2+ (Top Boxes) and Cd2+ (Lower Boxes) uptake in HEK293-T cells transfected with ZnT5 (Right Box) and control vector (Left Box). (E) ZnT5-mediated vesicular uptake of Zn2+ and Cd2+ normalized to control (mean ± s.e., n = 6). (*P ≤ 0.01).

Eitan Hoch, et al. Proc Natl Acad Sci U S A. 2012 May 8;109(19):7202-7207.
4.
Fig. 5.

Fig. 5. From: Histidine pairing at the metal transport site of mammalian ZnT transporters controls Zn2+ over Cd2+ selectivity.

Transport specificity of YiiP. (A) Kinetics of Zn2+ and Cd2+ transport for YiiP and YiiPD45H as indicated. The initial rate of Zn2+ or Cd2+ flux was plotted as a function of the metal concentration. The rates (filled circles) were fitted to a hyperbolic equation (solid line) to yield kinetic parameter Km,Vmax Vmax/Km. (B) YiiP-mediated metal uptake. Reconstituted proteoliposomes or control liposomes were briefly exposed to a panel of metal ions as indicated. Vesicles were washed free of extravesicular metal, and then subjected to ICP-MS quantification. The background metal levels were subtracted from the total metal readings to yield the net metal uptakes for proteoliposomes (closed bar, mean ± s.e., n = 4) and liposomes (open bars, mean ± s.e., n = 4). (*P ≤ 0.01).

Eitan Hoch, et al. Proc Natl Acad Sci U S A. 2012 May 8;109(19):7202-7207.
5.
Fig. 3.

Fig. 3. From: Histidine pairing at the metal transport site of mammalian ZnT transporters controls Zn2+ over Cd2+ selectivity.

A selectivity determinant residue for Zn2+ over Cd2+. (A) Fura-2 fluorescence in response to Cd2+ withdrawal as described in Fig  1B. The red and blue traces were recorded from HEK293-T cells expressing ZnT5 and ZnT5H451D (Top Box) and ZnT8 and ZnT8H106D (Lower Box), respectively. (B) Relative Zn2+ and Cd2+ efflux rate (mean ± s.e., n = 3) normalized to the rate recorded from HEK293-T cells transfected with control vector. (C) Zinpyr-1 fluorescence imaging of vesicular Cd2+ uptake in HEK293-T cells transfected with ZnT5 (Top Left Box) and ZnT5H451D (Top Right Box) or ZnT8 (Lower Left Box) and ZnT8H106D (Lower Right Box). (D) Vesicular Cd2+ uptake (mean ± s.e., n = 6) normalized to the total Cd2+ uptake measured from HEK293-T cells transfected with control vector. Note that a single H-to-D mutation in both ZnT5 and ZnT8 causes a loss of selectivity against Cd2+. (*P ≤ 0.01).

Eitan Hoch, et al. Proc Natl Acad Sci U S A. 2012 May 8;109(19):7202-7207.

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