Identification Of Nuclear Localization Signal Of Zebrafish Hypoxia-inducible Factor-3 Alpha

HIFs are crucial transcription factors mediating cellular response to hypoxia and play important roles in regulating kinds of genes in their mRNA levels. Due to hypoxia is a common physiological and pathological phenomenon in organisms, groups all over the world are interested in the researches on the protein structures, functions and the roles in some diseases of HIFs. HIFs are dimeric proteins composed of anα-subunit and aβ-subunit. Theα-subunit is an oxygen-dependent protein, which is crucial for the HIF complex. Three members of HIF-αhave been found, named HIF-1α, HIF-2αand HIF-3α, which are conserved in the protein structure. In hypoxia, HIF-αis stablised and enters into the nucleus to dimerize with HIF-1β. Then the HIF complex obtains its transcriptional activity and can regulate its target genes in the mRNA level.The reports on HIF-1αand HIF-2αreveal that HIF-αs, ranging from protein stability to function of transcriptional activity, are regulated by several mechanisms. Being transcription factors, whether the proteins are able to transport into the nucleus from the cytoplasm is also a precondition for the function of HIF-αs. As a result, it is necessary to find out the subcellular localization of HIF-α. Proteins larger than 50 kDa require a facilitated way to transport into the nucleus through nuclear pore complexes (NPC) unlike small molecules. A signal called nuclear localization signal (NLS) is involved in this transportation. According to previous reports, exogenous and endogenous HIF-1αor HIF-2αin the cells is detected in the nucleus. Moreover, the twoα-subunits are predominantly localized to the nucleus induced by hypoxia while in normoxia they appeared in the whole cell. In the protein structures, several groups have found their functional NLSs. The C-terminal of HIF-1αor HIF-2αcontains a fragment rich in basic amino acids (Lys and Arg). When these amino acids are deleted from the protein or substituted by other amino acids, both of the proteins lose the ability to localize in the nucleus. Compared to HIF-1αand HIF-2α, HIF-3αwas cloned later. Up to now, there is restricted information about this factor. For example, whether HIF-3αis a nuclear protein? Whether its localization is regulated by hypoxia? And if it can enter into the nucleus, where is the NLS contained in the protein? All of these questions need to be answered. Zebrafish Hif-3αis used in this research to identify its cytoplasm-nucleus distribution and its NLS(s). The identity of amino acids sequence between zebrafish Hif-3αand human HIF-3αis about 40%, suggesting they are relative conserved. Identification of the NLS in Hif-3αcan add more evidence in analyzing and comparing the commonalities and differences about protein structures among three HIF-αs.The full length of Hif-3α, its N-terminal or C-terminal amino acids sequences are all linked to EGFP, constructing several different fusion proteins. Then Hela cells are transfected with corresponding plasmids. Under normoxic or hypoxic condition, Hif-3α-EGFP is observed predominantly in the nucleus. When the C-terminal fragments are deleted from the entire protein, the ability to stay in the nucleus of the protein is obviously impared. However, lacking the N-terminal regions has no effect on its nuclear localization. These results indicate that the C-terminal of Hif-3αmay be functional as NLS. Further, a cytoplamic proteinβ-Gal-EGFP is used to map the NLS. The C-terminal of Hif-3α(457-626aa) is cut into several smaller fragments, which are linked toβ-Gal-EGFP. Transfection results show Hif-3α/518-567 and Hif-3α/568-626 can leadβ-Gal-EGFP into the nucleus. And four basic amino acids (K524,R525,K578 and R579) in the two sequences are essential for their function. Mutation of these amino acids in the full length protein also can decrease its nuclear distribution. Between the two basic motifs there is a 52 aa spacer. All of these amino acids compose a functional sequence which is necessary and sufficient for the nuclear localization of Hif-3α. Intriguingly, Hif-3α/1-69 also exhibits the ability to leadβ-Gal-EGFP into the nucleus and R14, K15, R25, R26 are important residues. However, this region can not work in the context of the full length protein, which is consistent with previous report in HIF-1αand HIF-2α. At last, upon sequence alignment using nine HIF-αs in their C-terminal, the NLS found in zebrafish is predicted a conserved NLS in HIF-α.In conclusion, zebrafish Hif-3αis a nuclear protein and its cellular distribution is not influenced by oxygen condition. Moreover, the protein contains a functional NLS in its C-terminal and four basic amino acids play necessary and sufficient roles in this distribution.