Research On The Mechanism Of Subcellular Localization Of Apoptin

Apoptin, a small protein derived from chicken anemia virus, causes apoptosis of chicken hemocytoblasts and precursor lymphocytes, leading to severe anemia and immunodeficiency. It also induces apoptosis in tumor and transformed cells, but not in normal cells. So, Apoptin is considered the first protein which can specifically induce apoptosis in tumor cells.The apoptosis-inducing activity of Apoptin is closely linked to its subcellular localization. In normal cells, Apoptin is localized predominantly in the cytoplasm, and fails to induce apoptosis, whereas in transformed and malignant cells, it is in the nucleus, and induces apoptosis. So it is very important to clarify the mechanism of its different subcellular localization in different cells.To study the mechanism of subcellular localiztion of Apoptin, we firstly observed the subcellular localization of enhanced green fluorescence protein-tagged Apoptin (Apoptin-EGFP) in various kinds of tumor or normal cells. The results indicated that Apoptin-EGFP predominantly distributes in cytoplasm in normal cells, whereas in tumor cells investigated, it mainly accumulates in the nucleus, and exists as globular aggregates, but in some tumor cells in M phase, it is found in cytoplasm. This results show that EGFP can be fused to Apoptin without affecting its subcellular localization.To discern why Apoptin has the ability to translocate to nuleus in tumor cells, the nuclear localization signal(NLS) was predicted, and then a series of NLS mutants were constructed. The subcellular localization of the NLS mutants tagged by EGFP was analyzed by fluorescence microscopy. The results indicated that Apoptin has no functional monopartite NLS, but has a bipartite NLS. It is the bipartite NLS that makes translocation of Apoptin to the nucleus of tumor cells. Further studies demonstrated that nuclear translocation of Apoptin in tumor cells is not regulated by phosphorylation. The other mechanism for the regulation of nuclear translocation of Apoptin must exist.A putative nuclear export signal (pNES) sequence was found in the C terminus of Apoptin. LMB, an inhibitor for exportin-1-dependent nuclear export, was used to analyze the function of pNES. It is found that in LMB treated primary rat hepatocytes, Apoptin still accumulates in cytoplasm. This means that the pNES of Apoptin is not a exportin-1-dependent export signal.A pNES deletion mutant of Apoptin was used to transfect the primary rat hepatocytes. 24h later, the mutant mainly accumulated in nucleus, whereas the wildtype of Apoptin predominantly distributed in cytoplasm. The results indicated that the pNES is responsible for the cytoplamic distrubution of Apoptin in normal cells. The results also implied that the NLS of Apoptin is not a tumor cell-specific NLS. The tumor cell-specific nuclear translocation of Apoptin depends on the simultaneous presence of its NLS and pNES.Further studies demonstrated the cytoplasmic retention of Apoptin in normal cells is controlled by the balance between the cytoplasmic retention and nuclear export. When pNES was deleted or heterogeneous NLS was introduced into Apoptin, the balance was disturbed, Apoptin was effectively translocated to the nucleus of normal cells. This implied that the cytoplasmic retention of Apoptin in normal is not absolute.On the other hand, it was found that the pNES of Apoptin is responsible for its mutimerization. The pNES deletion mutant of Apoptin seldom formed visible aggregates in normal or tumor cells, and when it was expressed in E.coli, it mainly existed in soluble form. In contrast, the wild type of Apoptin expressed E.coli formed inclusion bodies, and it is very difficult to denature and renature. As we know, the tendency of aggregation is related to formation of inclusion body in E.coli. This suggested that pNES contributes to aggregation of Apoptin. Recombinant pNES deletion mutant of Apoptin expressed in E.coli existed in monomer, whereas the wild type of the refolded Apoptin could form dimmer. Taken together, it could be concluded that pNES is neces...