Mobility within the nucleus and neighboring cytosol is a key feature of prothymosin-alpha

Prothymosin alpha is a small, unfolded, negatively charged, poorly antigeni c mammalian protein with a potent nuclear localization signal. Although it is apparently essential for growth, its precise function is unknown. We exa mined the location and behavior of the protein bearing different epitope ta gs using in situ immunolocalization in COS-1 and NIH3T3 cells. Tagged proth ymosin or appeared to be punctate and widely dispersed throughout the nucle us, with the exception of the nucleolus. A tiny cytoplasmic component, whic h persisted in the presence of cycloheximide and actinomycin D during inter phase, became pronounced immediately before, during, and after mitosis. Whe n nuclear uptake was abrogated, small tagged prothymosin or molecules, but not prothymosin alpha fused to beta-galactosidase, accumulated significantl y in the cytoplasm. Tagged prothymosin or shared domains with mobile protei ns such as Ran, transportin, and karyopherin beta, which also traverse the nuclear membrane, and co-localized with active RNA polymerase II. Mild digi tonin treatment resulted in nuclei devoid of prothymosin alpha. The data do not support tight binding to any nuclear component, Therefore, we propose that prothymosin alpha is a highly diffusible bolus of salt and infer that it facilitates movement of charged molecules in highly charged environments within and near the nucleus.