Mammalian karyopherin alpha 1 beta and alpha 2 beta heterodimers: alpha 1 or alpha 2 subunit binds nuclear localization signal and beta subunit interacts with peptide repeat-containing nucleoporins.

Abstract

Although only 44% identical to human karyopherin a,, human karyopherin a2 (Rchl protein) substituted for human karyopherin a, ( (3, 4). Indeed, mapping of the docking site(s) for Nup98 showed it (them) to be located in the repeat-containing N-terminal half of Nup98 (12). Finally, the repeat-containing nucleoporins also contain binding sites for plO (M. Matunis, G.B., and M.H., unpublished data). It has been proposed that the repeatcontaining nucleoporins serve as a stationary phase and the transport factors as the mobile phase in transport across the NPC (12).We showed previously that the a subunit serves as an NLS receptor and proposed that the X3 subunit serves as an adaptor that binds to the a subunit-NLS substrate complex and to the repeat-containing nucleoporins (4). Here we show that the previously used karyopherin a subunit [corresponding to hSRP-1/NPI-1 (14, 15)], now termed karyopherin a1, can be replaced by karyopherin a2 [corresponding to the hSRP-1-related Rchl (16)]. Although karyopherin a, and a2 are only 44% identical, we did not detect any functional difference. We also show that karyopherin 3 is localized in the cytoplasm and at the nuclear rim and is largely lost after digitonin permeabilization of cells. Unlike karyopherin a, which alone cannot bind to the nuclear envelope of digitonin-permeabilized cells, karyopherin , can bind without karyopherin a being present. In an import reaction with all recombinant transport factors, the karyopherin a subunits, Ran, and plO are shown to enter the nucleus, whereas the ,B subunit remains at the nuclear envelope. This result suggests that the heterodimeric karyopherin complex is dissociated during transport. Overlay binding assays showed that the ,B subunit binds to the repeatcontaining nucleoporins. These data are consistent with the previously proposed function of karyopherin J3 as an adaptor between karyopherin a/NLS substrate complex and repeatcontaining nucleoporins.Abbreviations: HSA, human serum albumin; NLS, nuclear localization sequence; NPC, nuclear pore complex; FITC, fluorescein isothiocyanate.

Protein import into the nucleus

“…Similarly, incubation of immobilized GLFG repeats of Nup98 with a cytosolic subfraction led to depletion of the binding activity [47]; NRI3 was identified as one protein bound to these repeats [12]. Binding of the NLS-receptor to some but not all FG nucleoporins has also been reported by others [20,32,45,[48][49][50] and required only the repeat domains [32,45,49,50].…”

“…NRc~ is found in a complex with NRI3 [17,12,13,18,19]. Both subunits are required for binding and transport [17,20]. NRa from yeast, also termed Srplp or Kap60p [18,21], is most likely identical to the previously characterized NLS-binding protein NBP70 [22,23].…”

“…NRa from yeast, also termed Srplp or Kap60p [18,21], is most likely identical to the previously characterized NLS-binding protein NBP70 [22,23]. While there is only one yeast gene encoding NRa, higher eukaryotes contain a group of similar genes, which suggests differences in substrate specificity [11,20,24-281. NRc~ binds directly to nuclear localization sequences [14,22,17,20,26,29,30]. NR[3 was found to have weak [17] or no NLS-binding activity by itself [31,32] but it cooperates with NRc~ in NLS-recognition and binding of import substrate to the NE [16,17,32].…”

“…The NR heterodimer was observed to bind import substrates in cytosolic extracts [12,13,17]. The NR/substrate complex docks via NR[~ to the NPC [20,35] and is translocated through the NPC by an energy-dependent and Ran-dependent mechanism. The aminoterminal ,~ 50 amino acids of NRct are necessary and sufficient for NR [3 binding.…”

“…Yeast NRct was localized to the nucleus, or the NE, or the cytoplasm [21,22,[37][38][39]. NR[3 is located predominantly at the NE, also in the cytoplasm but not in the nucleoplasm [16,20,39]. Immunoelectron microscopy revealed that NR~ is located on both sides of the NPC [35].…”