RESUMEN
The authors studied inclusion formation in vitro using transiently transfected PC12 cells, with epitope-tagged and untagged full-length and truncated wild-type and expanded ataxins -1, -2, -3, and -7. At 72 hours, no inclusions were seen with wild-type full-length or truncated ataxins -2, -3, or -7, and only one with ataxin-1. Truncation abolished nuclear localization of ataxins -1 and -7, and allowed nuclear entry of ataxin-2. Of the expanded ataxins, only -1 and -2 formed inclusions, and those of ataxin-2 were rare and exclusively cytoplasmic. Truncation resulted in inclusion formation by ataxins -3 and -7, increased ataxin-1 inclusions, and enabled formation of nuclear ataxin-2 inclusions. There was no recruitment of wild-type ataxin-1 to expanded ataxin-1 inclusions.
Asunto(s)
Cuerpos de Inclusión/metabolismo , Proteínas del Tejido Nervioso/fisiología , Animales , Ataxina-1 , Ataxina-3 , Ataxina-7 , Ataxinas , Mutación/fisiología , Proteínas del Tejido Nervioso/genética , Proteínas Nucleares/genética , Proteínas Nucleares/fisiología , Células PC12 , Ratas , Transfección/métodosRESUMEN
We have developed a monoclonal antibody (4A7) directed against the C-terminus of the ataxin-2 protein that is involved in the polyglutamine neurodegenerative disorder spinocerebellar ataxia type 2. Comparison with other ataxin-2 antibodies showed that 4A7 specifically recognized ataxin-2. In contrast, a previously reported ataxin-2 antibody (15F6) did not appear to recognize full-length ataxin-2 in our systems. Immunocytochemical and subcellular fractionation studies using 4A7 confirmed previous reports that ataxin-2 is localized to both the cytoplasm and the trans-Golgi network in rat PC12 cells and rat brain tissue. In contrast, 4A7 failed to label the trans-Golgi network in the three primate cell lines examined. Cytoplasmic ataxin-2 was not associated with mitochondria, lysosomes, endoplasmic reticulum, peroxisomes, proteasomes, clathrin-coated pits or vesicles, or F-actin. Ataxin-2 was found to be phosphorylated but not glycosylated, and exhibited an estimated half-life of not less than 21 h. Interestingly, another commercially available ataxin-2 antibody did not detect ataxin-2 localized to the trans-Golgi network. This antibody was also found to immunoprecipitate fewer proteins/protein partners than 4A7. Although cross-reactivity of the 4A7 antibody with other protein(s) cannot be ruled out, it appears likely that the interaction of ataxin-2 with other cell components is dependent on both the host cell type and its subsequent subcellular localization.
