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Y. Gribanova, N. B. Akhmedov, E. Mendoza, C. K. Yamashita, J. E. Johnson, D. A. Fox, D. B. Farber; The Role of the Mouse Retinal 7R Protein in Maintaining the Golgi Structure. Invest. Ophthalmol. Vis. Sci. 2008;49(13):152.
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To evaluate the relative distribution of the 7R protein in cultured cells and mouse retina and to determine the effect that 7R has on the Golgi structure in retinal cell types.
HEK293 cells were transfected with a pcDNA4/HisMax/7R construct containing the open-reading frame of 7R fused to His and Xpress tags. Immunohistochemistry was performed with 7Rc, anti-Xpress and various retinal antibodies to localize the compartmental distribution of 7R in transfected and nontransfected cells as well as in mouse retinal sections.
We have previously detected the juxtanuclear localization of 7R protein in the ER/Golgi apparatus region of transfected HEK 293 cells. Immunoblots of retinal proteins from sucrose gradient fractions confirmed that a significant amount of 7R protein is found in the Golgi membrane complex. To corroborate that indeed 7R is a Golgi- localized protein, the 7Rc antibody as well as antibodies to two markers of the cis-Golgi complex, GM130 and GS28, were used in immunofluorescence microscopy experiments. As expected, 7R was observed in the perinuclear structures co-staining with Golgi markers. As an alternative approach to evaluating the Golgi localization of 7R, we also tested its sensitivity to brefeldin A (BFA), a Golgi-destabilizing agent. The treatment of transfected HEK 293 cells with BFA resulted in disruption of the stacked Golgi structure and a rapid dissociation of 7R from Golgi membranes, as revealed by a diffuse cytoplasmic staining. Interestingly, we also noticed that the structure of the Golgi complex was altered and tended to be more compact in cells overexpressing 7R, whereas untransfected cells had Golgi complexes with normal crescent-shaped reticular morphology that followed the contour of the nucleus. Immunohistochemical labeling of retinal sections with the 7Rc antibody showed localization in the perinuclear region of photoreceptors, in all cell types of the inner nuclear layer and in perinuclear region of ganglion cells. In addition, 7Rc intensely labeled cone outer segments and Muller cells.
7R is localized mostly to the cis-Golgi membranes of cells and associates with the Golgi apparatus in a BFA-sensitive manner. A significant compaction of the Golgi complex was observed only in cells overexpressing 7R. We interpret these results as an indication that at least one function of 7R protein is to maintain Golgi structure. Also, the 7R protein may be involved in cone disk membranes’ organization.
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