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First published online 6 September 2005
doi: 10.1242/jcs.02570

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Subcellular localization of iron regulatory proteins to Golgi and ER membranes

¹ Department of Neurosurgery, G.M. Leader Family Laboratory for Alzheimer's Disease Research, Pennsylvania State University College of Medicine, Hershey, PA 17033, USA
² New York University, Department of Nutrition and Food Studies, 35 West 4th Street, 10th floor, New York, NY 10012-1172, USA
³ Department of Nutritional Sciences, Pennsylvania State University, S-126 Henderson Building, University Park, PA 16802, USA

View larger version (70K): [in a new window]   Fig. 1. Cellular distribution of IRP1 and IRP2. (A,B) Distribution of (A) IRP1 using an IRP1 antibody (red) and (B) IRP2 using an IRP2 antibody (red), in NIH3T3 cells. The cell nuclei are visualized with DAPI (blue). Both A and B reveal a cytosolic distribution for IRP1 and IRP2 but IRP2 distribution is more punctate than IRP1. (C,D) Distribution of (C) c-myc-tagged IRP1 and (D) c-myc-tagged IRP2, in NIH3T3 cells. C and D are representative cells that show the cytosolic distribution of both IRP1 and IRP2 (clear area is the cell nucleus). IRP1 is localized more in the perinuclear region whereas IRP2 localization extends further toward the perimeter of the cell.

View larger version (90K): [in a new window]   Fig. 2. Co-localization of IRP1 with the ER marker calnexin in SW1088 cells. (A-C) Representative cells selected from D (arrow). A-C are confocal images of the same cells showing the intracellular distribution of IRP1 (green in A), the ER marker calnexin (red in B) and the merged image (C). The yellow indicates strong overlap in the perinuclear region.

View larger version (73K): [in a new window]   Fig. 3. Co-localization of IRP1 with the Golgi marker 58K Golgi protein in SW1088 cells. A-C are representative cells selected from D (arrow). A-C are confocal images of the same cells showing the intracellular distribution of IRP1 (green in A), the Golgi marker Golgi 58K protein (red in B) and the merged image (C). The yellow indicates strong overlap in the perinuclear region.

View larger version (64K): [in a new window]   Fig. 4. Co-localization of IRP1 with the ER marker calnexin in iron-depleted or iron-loaded SW1088 cells. These are representative confocal images of SW1088 cells that were exposed to iron (A-D) or to the iron chelator DFO (E-H) for 72 hours prior to fixation. Following fixation the cells were stained with DAPI (blue) to visualize the nucleus and with an IRP1 antibody (green in B and F) or the ER marker calnexin (red in C and G). The merged images are shown in D and H. The co-localization of IRP1 and the ER are indicated by yellow. Treatment with iron or an iron chelator did not result in changes in IRP1 distribution that could be identified microscopically.

View larger version (25K): [in a new window]   Fig. 5. Distribution of ER in density gradient fractions in relation to IRP1 immunoreactivity. This graph shows the distribution of IRP1 and ER immunoreactivity in control rat livers. The primary y-axis shows the IRP1 and ER distribution in the density gradient fractions. The secondary y-axis shows the ER marker, cytochrome C-NADPH reductase, which was used to plot the distribution of ER in fractions obtained from density gradient centrifugation. The fraction number is shown on the x-axis. Enzyme activity in each fraction is plotted as a function of total protein. 5 µg of each fraction was subjected to western blot analysis to determine IRP1 immunoreactivity. These data confirm the localization of IRP1 immunoreactivity in ER containing fractions. These experiments were performed in duplicate and representative data are shown.

View larger version (22K): [in a new window]   Fig. 6. Representative IRP1 subcellular distribution from control rat livers. This graph shows the distribution of IRP1, ER and Golgi immunoreactivity in liver fractions from rats fed a normal diet. The y-axis shows the mean pixel density for IRP1, ER and Golgi in the density gradient fractions. The fraction number is shown on the x-axis. Total protein (10 µg) was added to detect IRP1 and 5 µg of protein was added to detect both ER and Golgi by western blot analysis. These data demonstrate that under control conditions, a large proportion of IRP1 immunoreactivity is found in cytosol-containing fractions. Additionally, under control conditions, a small proportion of IRP1 immunoreactivity is also found in Golgi-containing fractions. These experiments were run in triplicate and representative data are shown.

View larger version (26K): [in a new window]   Fig. 7. Representative IRP1 subcellular distribution from iron-depleted rat livers. This graph shows the distribution of IRP1, ER and Golgi immunoreactivity in liver fractions from rats fed a low-iron diet. The y-axis shows the mean pixel density for IRP1, ER and Golgi in the density gradient fractions. The fraction number is shown on the x-axis. Total protein (10 µg) was added to detect IRP1 and 5 µg of protein was added to detect both ER and Golgi by western blot analysis. These data indicate that under iron-depleted conditions, the IRP1 immunoreactivity is found in ER-containing fractions as well as fractions that contain Golgi immunoreactivity. These experiments were run in triplicate and representative data are shown.

View larger version (23K): [in a new window]   Fig. 8. Electrophoretic mobility shift assays (EMSAs) for IRPs in HEK-293 cells in response to iron loading (FAC), iron chelation (DFO), treatment with hydrogen peroxide (100 µM for 30 minutes), and IL-1ß (10 ng/ml for 16 hours). The IRP/IRE interaction decreases with iron and IL-1ß treatments in both the cytosolic and membrane fractions, and IRP/IRE interaction increases with DFO and hydrogen peroxide treatments in both the cytosolic and membrane fractions. The data suggest IRPs localize to the cytosolic fraction with iron treatment and localize to the membrane fraction with H2O2 treatment. *P<0.05.

View larger version (17K): [in a new window]   Fig. 9. Electrophoretic mobility shift assays (EMSAs) for IRPs in SW1088 cells in response to phorbol 12-myristate 13-acetate (PMA, 0.2 µM) and chelerythrine (CET, 2.0 µM). IRP/IRE interaction in SW1088 cells decreases upon treatment with PMA in both the cytosolic and membrane fractions and increases upon treatment with CET in both the cytosolic and membrane fractions. The data suggest IRP localization to the cytosolic fraction occurs with protein kinase C inhibition. *P<0.05.

View larger version (30K): [in a new window]   Fig. 10. Electrophoretic mobility shift assays (EMSAs) for IRPs in SW1088 cells in response to iron exposure (FAC), iron chelation (DFO), calcium chelation (BAPTA), and BAPTA concomitant with FAC (B-FAC) or DFO (B-DFO). IRP/IRE interaction in SW1088 cells decreases upon FAC treatment with or without concomitant addition of the calcium chelator, BAPTA in both the cytosolic and membrane fractions. The IRP/IRE interaction increases upon treatment with DFO alone, BAPTA alone or DFO/BAPTA together in both the cytosolic and membrane fractions. IRP localization to the membrane fraction occurs with iron and calcium chelation. *P<0.05.

View larger version (19K): [in a new window]   Fig. 11. Proposed mechanism for IRP1 interaction with the ER and Golgi membrane fractions. This schematic demonstrates the intracellular distributions of IRP1 discovered in this study. IRP1 is found in Golgi membranes in addition to the cytosol. Under conditions of low iron and in cells in culture, IRP1 is also found in the ER membranes. We propose that under low iron conditions IRP1 is incorporated into the ER membrane by a hydrophobic tail immediately after translation. Within the ER, IRP1 can serve to further stabilize transferrin receptor (TfR) mRNA by binding to IREs that were not bound in the cytoplasm. Once the TfR mRNA has been translated, the IRPs in the ER membrane move to the Golgi membrane where they can receive an Fe-S cluster. The acquisition of an Fe-S cluster is not required for IRP1 to be released from the Golgi membrane to the cytosol or be recycled to the ER membrane.