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N. B. Akhmedov, E. Mendoza, C. K. Yamashita, M. Saghizadeh, Y. Gribanova, D. B. Farber; A Novel Protein of the Mammalian Retina, 7R Is Associated With the Golgi Complex. Invest. Ophthalmol. Vis. Sci. 2007;48(13):3059.
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The objective of the current study was to characterize the 7R gene and its gene product, to examine the distribution of the 7R protein in the retina and to determine its cellular function.
Immunohistochemical studies were performed to localize the 7R protein in transfected cell lines and mouse retinal tissue sections. The polyclonal peptide antiserum, R7c, generated against the C-terminal region (18 aa) of 7R was used to examine the retinal expression pattern of the 7R protein. To determine the compartmental distribution of 7R, the post-nuclear supernatants were fractionated on two sequential discontinuous sucrose gradients in a Beckman TL100 ultracentrifuge.
The 7R gene is predicted to encode a protein with seven transmembrane domains. Our studies revealed that the mRNA corresponding to 7R is present in most tissues as a 2.0 kb transcript. Transfection of an expression vector containing the open-reading frame of 7R fused with His and anti-Xpress tags at the N-terminus into HEK 293 cells followed by immunocytochemistry with anti-Xpress antibody revealed the cytoplasmic localization of the 7R protein. The endogenous 7R was also localized to the cytoplasm of HEK cells with the polyclonal anti-R7c peptide antibody. In addition, all three nuclear layers of normal mouse retinal sections were heavily labeled with the R7c antibody and confirmed the cytoplasmic nature of 7R. This specific staining was inhibited by preincubation of the antibody with the immunogenic peptides. More precise analysis of the 7R protein expression by confocal microscopy showed its juxtanuclear localization, corresponding to the ER/Golgi apparatus region. Immunoblots of retinal sucrose gradient fractions with anti-R7c and CM130 antibodies also showed that a significant amount of 7R protein was found in the Golgi membrane complex.
7R is an ubiquitously expressed protein of unknown function. Its perinuclear expression pattern and the results of the cellular fractionation experiments showed that 7R is located in stacked Golgi membranes. Thus, 7R may be an integral component of the Golgi apparatus and may play a role in Golgi function.
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