Mobility within the nucleus and neighboring cytosol is a key feature of prothymosin-alpha.
Prothymosin alpha is a small, unfolded, negatively charged, poorly antigenic mammalian protein with a potent nuclear localization signal. Although it is apparently essential for growth, its precise function is unknown. We examined the location and behavior of the protein bearing different epitope tags using in situ immunolocalization in COS-1 and NIH3T3 cells. Tagged prothymosin alpha appeared to be punctate and widely dispersed throughout the nucleus, with the exception of the nucleolus. A tiny cytoplasmic component, which persisted in the presence of cycloheximide and actinomycin D during interphase, became pronounced immediately before, during, and after mitosis. When nuclear uptake was abrogated, small tagged prothymosin alpha molecules, but not prothymosin alpha fused to beta-galactosidase, accumulated significantly in the cytoplasm. Tagged prothymosin alpha shared domains with mobile proteins such as Ran, transportin, and karyopherin beta, which also traverse the nuclear membrane, and co-localized with active RNA polymerase II. Mild digitonin 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.
Enkemann
S A
SA
Section on Genes and Gene Products, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, USA.
Ward
R D
RD
Berger
S L
SL
D016475:3T3 Cells; D000818:Animals; D000906:Antibodies; D019556:COS Cells; D002467:Cell Nucleus / Q000378:metabolism*; D002522:Chlorocebus aethiops; D003513:Cycloheximide / Q000494:pharmacology; D003600:Cytosol / Q000378:metabolism*; D003609:Dactinomycin / Q000494:pharmacology; D004058:Diffusion; D004072:Digitonin / Q000494:pharmacology; D000939:Epitopes / Q000276:immunology; D007399:Interphase; D051379:Mice; D008938:Mitosis; D011498:Protein Precursors / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D013947:Thymosin / Q000031:analogs & derivatives* / Q000037:antagonists & inhibitors / Q000378:metabolism*
D000906:Antibodies; D000939:Epitopes; D011498:Protein Precursors; D011500:Protein Synthesis Inhibitors; C040782:prothymosin alpha; D003609:Dactinomycin; D013947:Thymosin; D003513:Cycloheximide; D004072:Digitonin