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. 2003 Feb 4;100(3):1215-20.
doi: 10.1073/pnas.0336230100. Epub 2003 Jan 21.

Essential role for Atox1 in the copper-mediated intracellular trafficking of the Menkes ATPase

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VSports在线直播 - Essential role for Atox1 in the copper-mediated intracellular trafficking of the Menkes ATPase

Iqbal Hamza et al. Proc Natl Acad Sci U S A. .

Abstract (V体育官网)

The metallochaperone Atox1 directly interacts with the copper-transporting ATPases and plays a critical role in perinatal copper homeostasis. To determine the cell biological mechanisms of Atox1 function, intracellular copper metabolism, and Menkes ATPase abundance, localization and trafficking were examined in immortalized fibroblast cell lines derived from Atox1(+/+) and Atox1(-/-) embryos. Consistent with the proposed role for Atox1 in copper delivery to the secretory pathway, a marked increase in intracellular copper content secondary to impaired copper efflux was observed in Atox1-deficient cells. Although the localization of the Menkes ATPase was identical in Atox1(+/+) and Atox1(-/-) cells under conditions of equivalent intracellular copper content, a significant impairment in copper-mediated Menkes ATPase trafficking was observed in the absence of Atox1 VSports手机版. When quantitative confocal immunofluorescence was used, significant differences in the time and dose-dependent trafficking of the Menkes ATPase from the Golgi compartment in response to copper were observed between Atox1(+/+) and Atox1(-/-) cells. These data reveal an essential role for Atox1 in establishing the threshold for copper-dependent movement of the copper-transporting ATPases within the secretory compartment and that, in the absence of Atox1, this movement alone is not sufficient to restore normal copper efflux. Taken together, these findings provide a cell biological model for the role of this metallochaperone under the physiological conditions of copper limitation in mammalian cells. .

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Figures (V体育官网入口)

Figure 1
Figure 1
Characterization of Atox1-deficient cells. (A) Immunoblot analysis of SV40 large T antigen in primary (P) and immortalized (I) embryonic fibroblasts. Cell lysate (100 μg) was separated by 4–20% SDS/PAGE and analyzed with SV40 T antigen antisera followed by chemiluminescent detection. Cos7 cells express T antigen and were used as a positive control. (B) Copper retention was determined in cultured Atox1+/+ and Atox1−/− cells, and cell lines were derived from Mobr-J mice (802-1Mnk) or the corresponding wild-type control (802-5). A total of 2 × 105 cells were incubated with 8 × 106 cpm of 64Cu. After 68 h, the cells were washed, lysed, and analyzed for 64Cu retained by using a γ counter. Each data point represents the mean ± SEM from three separate experiments performed in triplicate and expressed as cpm/μg of total cell protein (*, P < 0.05). (C) Immunoblot analysis of Atox1 protein in mouse fibroblasts. Cell lysate (100 μg) was separated by 10–20% Tricine SDS/PAGE and probed with Atox1 antisera followed by chemiluminescent detection. This immunoblot was stripped to remove Atox1 antibodies and reprobed with SOD1 antisera (D). SOD1 migrates slower than the expected 16 kDa on Tricine SDS/PAGE used to analyze low molecular weight proteins such as Atox1.
Figure 2
Figure 2
Immunolocalization of Menkes ATPase in Atox1-deficient cells. (A) Immunoblot analysis of Menkes protein in mouse fibroblasts. Cell lysate (100 μg) was separated by 4–20% SDS/PAGE and probed with Menkes antisera (1:2,000) generated against the C terminus of the Menkes ATPase, followed by chemiluminescent detection. Less than 2% of Menkes protein was detected in 802-1 cells compared with wild-type 802-5 cells. (B) Double label indirect immunofluorescence localization (×63) in 802-5 and 802-1 cells grown in basal media, fixed, and stained with GS28 (α-GS28, Alexa 488) and Menkes (α-Mnk, Alexa 546) antibodies, and analyzed by using epifluorescence microscopy. Images of Menkes and GS28 Golgi marker are merged to depict overlapping regions. (Scale bar, 50 μm.) (C and D) Immortalized Atox1+/+ and Atox1−/− cell lines were grown in basal media (C) or in the presence of 200 μM BCS (D) and processed for indirect immunofluorescence studies as described above by using epifluorescence microscopy. (Scale bar, 50 μm.)
Figure 3
Figure 3
Characterization of copper-mediated trafficking of Menkes protein. (A) Immortalized Atox1+/+ and Atox1−/− cells were grown on coverslips in the presence of 200 μM BCS for 48 h, followed by the addition of 0.5 or 10 μM CuCl2 for 1 h to the culture media, and processed for double-label immunofluorescence by using Menkes and GS28 antibodies for analysis with confocal microscopy. Images of Menkes and GS28 are merged to depict overlapping regions. (Scale bar, 50 μm.) (B) Quantitative analysis of Menkes signal in the Golgi was measured in immortalized Atox1+/+ cells by using double-label immunofluorescence studies as described in Materials and Methods. Cells were grown in the presence of 200 μM BCS followed by copper treatment for 3 h, and processed for immunostaining. Overlapping signal intensities were analyzed and quantitated by using a laser scanning confocal Olympus microscope and FLUOVIEW FV500 Version 3.3 program. Each data point represents the mean ± SEM from four separate experiments with a minimum of seven scans per experiment per data point. Each scan is comprised of 15–18 cells or at least 105 cells. (Inset) The same analysis plotted onto a logarithmic x axis (μM) for clarity.
Figure 4
Figure 4
Time- and dose-dependent trafficking of the Menkes protein. Quantitative immunofluorescence analysis of Menkes signal in the Golgi was measured in immortalized Atox1+/+ and Atox1−/− cells in 200 μM BCS for 48 h, followed by 1-, 2-, and 3-h incubations in the presence of 0.5 μM (A), 10 μM (B), or 100 μM (C) CuCl2 in the culture media. Overlapping signal intensities were analyzed and quantitated by using FLUOVIEW FV500 Version 3.3 program. Each data point represents the mean ± SEM from four separate experiments with a minimum of seven scans per experiment per data point. Each scan is comprised of 15–18 cells or at least 105 cells (*, P < 0.001 and #, P < 0.05 between Atox1+/+ and Atox1−/− cells). Mean values were calculated, and differences were compared by using a one-way ANOVA with a Student–Newman–Keuls multiple comparison test.

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