Abstract
Little quantitative, kinetic information is available with respect to the process of nuclear import of conventional nuclear localization sequence (NLS)-containing proteins, which initially involves recognition and docking at the nuclear pore by importin alpha/beta. This study compares the binding and nuclear import properties of mouse (m) and yeast (y) importin (IMP) subunits with respect to the NLSs from the SV40 large tumor antigen (T-ag), and the Xenopus laevis phosphoprotein N1N2. m- and y-IMPalpha recognized both NLSs, with y-IMPalpha exhibiting higher affinity. m-IMPbeta greatly enhanced the binding of m-IMPalpha to the T-ag and N1N2 NLSs, but y-IMPbeta did not significantly affect the affinity of y-IMPalpha for the T-ag NLS. In contrast, y-IMPbeta enhanced y-IMPalpha binding to the NLS of N1N2, but to a lesser extent than the enhancement of m-IMPalpha binding by m-IMPbeta. NLS-dependent nuclear import was reconstituted in vitro using the different importin subunits together with the transport factors Ran and NTF2. Whereas T-ag NLS-mediated nuclear import did not exhibit an absolute requirement for NTF2, N1N2 NLS-mediated transport strictly required NTF2. High levels of NTF2 inhibited nuclear accumulation conferred by both NLSs. We conclude that different NLSs possess distinct nuclear import properties due to differences in recognition by importin and requirements for NTF2.
Highlights
Little quantitative, kinetic information is available with respect to the process of nuclear import of conventional nuclear localization sequence (NLS)-containing proteins, which initially involves recognition and docking at the nuclear pore by importin ␣/
This study compares the binding and nuclear import properties of mouse (m) and yeast (y) importin (IMP) subunits with respect to the NLSs from the SV40 large tumor antigen (T-ag), and the Xenopus laevis phosphoprotein N1N2. mand y-IMP␣ recognized both NLSs, with y-IMP␣ exhibiting higher affinity. m-IMP greatly enhanced the binding of m-IMP␣ to the T-ag and N1N2 NLSs, but y-IMP did not significantly affect the affinity of y-IMP␣ for the T-ag NLS
We find that mouse (m-IMP␣) and yeast (y-IMP␣) importin ␣s can bind different types of NLS, y-IMP␣ binds with higher affinity. y-IMP does not significantly affect the affinity of y-IMP␣ for the NLS of the simian virus SV40 large tumor antigen (T-ag) but does enhance binding to the NLS of the Xenopus laevis phosphoprotein N1N2
Summary
Constructs to express fusion proteins T-ag-CcN--Gal, and the NLS-defective mutant derivative T-ag-Cc--Gal containing T-ag sequences fused amino-terminal to the Escherichia coli -galactosidase sequence (amino acids 9 –1023) have been described [30, 31]. The plasmid pPR-N1N2--Gal expressing Xenopus laevis N1N2 amino acids 465–581 fused amino-terminal to -galactosidase has been described [32]. M-IMP␣ (PTAC58), m-IMP (PTAC97), y-IMP␣ (Kap60), y-IMP (Kap95) subunits, and human Ran were expressed as glutathione S-transferase (GST) fusion proteins and purified as described [9, 21, 28, 34]. Fusion proteins contain the indicated amino acid sequences fused amino-terminal to E. coli -galactosidase (amino acid 9 —1023) The single letter amino acid code is used with small letters indicating variation from the wild type sequences [33]
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