Neurofibromin Colocalizes with Mitochondria in Cultured Cells

• Published in: Experimental cell research 1997-10, Vol.236 (1), p.161-172
• Main Authors: Roudebush, Margaret, Slabe, Tom, Sundaram, Vidya, Hoppel, Charles L., Golubic, Mladen, Stacey, Dennis W.
• Format: Article
• Language: English
• Subjects: 3T3 Cells - chemistry; 3T3 Cells - metabolism; Animals; Antibody Specificity; Astrocytoma; Binding, Competitive - immunology; Blotting, Western; Cytoskeleton - chemistry; Cytoskeleton - immunology; Glycogen Synthase - immunology; Humans; Immunoglobulin G - pharmacology; Liver Cirrhosis, Biliary - immunology; Mice; Mice, Knockout; Mitochondria - chemistry; Neurilemmoma; Neurofibromin 1; Peptide Fragments - immunology; Protein Kinase C - immunology; Proteins - analysis; Proteins - genetics; Proteins - immunology; Rats; Staining and Labeling; Tissue Fixation
• ISSN: 0014-4827; 1090-2422
• DOI: 10.1006/excr.1997.3712

Mutations in neurofibromatosis type 1 target the gene coding for neurofibromin. While neurofibromin is able to accelerate the rate of GTP hydrolysis by cellular Ras proteins, its biological function is not well understood. To gain information regarding its function, the intracellular localization of neurofibromin was analyzed in cultured cell lines using polyclonal antisera raised against four neurofibromin-specific peptides, three from the carboxyl terminus and one from the amino terminus. In methanol-fixed cells distinct rod-like structures distributed throughout the cytoplasm were recognized by the antisera. Similar structures were seen with each antiserum, including affinity-purified antibodies, and in each of the cultured cell lines tested. Similar structures were seen in paraformaldehyde-fixed cells. Double staining experiments showed that these structures colocalize with mitochondria, but not with actin, β-tubulin, or endoplasmic reticulum. When actin or tubulin structures within the cell were disrupted by separate antimitotic drugs, these stained structures retained their shape. Neurofibromin association with mitochondria was confirmed biochemically when highly purified mitochondrial fractions from bovine heart tissue were shown in Western analysis to contain neurofibromin. This association might be helpful in predicting identification of some of the cellular proteins with which neurofibromin interacts.