Protein import, nucleus nuclear localization signal

Fig. 10.22. Nuclear import. Proteins with the nuclear localization signal bind to importins, which carry them through the nuclear pore into the nucleus. The monomeric G protein Ran containing bound GTP binds to one of the subunits of importin. This causes dissociation of the importin subunits and release of the imported protein in the nucleus. The Ran-importin complex exits a nuclear pore. On the cytoplasmic side, a RanGAP (GTPase activating protein) activates the hydrolysis of GTP to GDP, which causes dissociation of the complex. RanGDP is subsequently returned to the nucleus, where an accessory protein activates dissociation of GDP and association of GTP.

Importins are transport proteins at the nuclear pore complex, needed for the selective import of proteins into the nucleus. They recognize nuclear localization signal sequences of cargo proteins. 

Sequence of amino acids that determine the transport of proteins into the nucleus. Although there is no clear consensus, nuclear localization signals tend to be rich in positively charged residues, which allow interaction with proteins from the nuclear import machinery (i.e., importins). 

Proteins destined for import into the nucleus typically require a nuclear localization signal, four to eight amino acids long, located internally in the protein. Uptake occurs via nuclear pores and requires ATP hydrolysis. 

Fig. 10.8 Above, import of the transcription factor NF-AT4 into the nucleus. In activated cells, import is initiated by calcineurin-mediated dephosphorylation of NF-AT4. Dephosphorylation unmasks the nuclear-localization signal (NLS), and at the same time blocks the nuclear export signal (NES). The NES is recognized by the exportin protein (Crml). Nuclear export is an active process. Moreover, nuclear export requires rephosphorylation of the NF-AT4 transcription factor. It is indicated that dephosphorylation by calcineurin and nuclear export are mutually exclusive, because calcineurin and Crm 1 compete for a common binding site on NES. When NES binds to Crml, NT-AT4 is exported from the nucleus, and when calcineurin binds to NES, NF-AT4 remains in the nucleus and forms a transcriptionally active complex. Below, how the extent of dephosphorylation controls the transcriptional activity of NF-AT4. When NF-AT4 is fully phosphorylated, NLS is hidden and the transcription factor remains in the cytoplasm. When NF-AT4 is only partially dephosphorylated, NLS is exposed and can interact with importin a/b which promote nuclear import, and at the same time, NES can interact with the exportin Crml, which promotes nuclear export. The consequence is that the transcription factor shuttles between the nucleus and the cytoplasm and is not transcriptionally active. In order to become transcriptionally fully active, NF-AT4 must be completely dephosphorylated. This prevents export from the nucleus by blocking NES, and may increase the affinity of the transcription factor for DNA by exposure of its trans-activating domain (TAD). (The entire scheme is reproduced with permission of Drs Patrick G. Hogan and Anjana Rao and Nature from Fig. 1 in ref. 68.)...

Proteins Imported to or exported from the nucleus contain a specific amino acid sequence that functions as a nuclear-localization signal (NLS) or a nuclear-export signal (NFS). Nucleus-restricted proteins contain an NLS but not an NES, whereas proteins that shuttle between the nucleus and cytoplasm contain both signals. 

In most multicellular eukaryotes, the nuclear envelope breaks down at each cell division, and once division is completed and the nuclear envelope reestablished, the dispersed nuclear proteins must be reimported. To allow this repeated nuclear importation, the signal sequence that targets a protein to the nucleus—the nuclear localization sequence, NLS—is not removed after the protein arrives at its destination. An NLS, unlike other signal sequences, may be located almost anywhere along the primary sequence of the protein. NLSs can vary considerably, but many consist of four to eight amino acid residues and include several consecutive basic (Arg or Lys) residues. 

Interestingly, the amino-terminal extensions contain putative nuclear localization sequences that appear to be involved in transport of these proteins into the nucleus (Acland et al., 1990 Amalric et al., 1991 Bugler et al., 1991 Kiefer et al., 1994). FGF-1 has also been shown to enter the nucleus, but the amino acid sequence involved in this process remains to be elucidated (Cao et al., 1993). The specific function of nuclear FGF is still unclear, but there is evidence that FGF-2 import into the nucleus is involved in the mitogenic response of endothelial cells to FGF (Bouche et al., 1987 Baldin et al., 1990). Such a nuclear mechanism of FGF action could supplement the classical signal pathway by receptor-mediated mechanisms. 

Das et al, showed that LdTOPlLS localize in both nucleus and kinetoplast of L. donovant (Fig. IB). The existence of multiple localization signals have been mappied in the larger subunits of Trypanosoma and Leishmania topoisomerase but no NLS has been found in smaller subunits of the enzyme. So it is likely that the subunits interact in the cytosol before nuclear and kinetoplast importation. But, whether the proteins perform separate functions in the cytoplasm is still unknown. 

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