Plant Importin α Binds Nuclear Localization Sequences with High Affinity and Can Mediate Nuclear Import Independent of Importin β

Abstract

Nuclear import of conventional nuclear localization sequence (NLS)-containing proteins initially involves recognition by the importin (IMP)␣The entry of karyophilic proteins into the nucleus through the nuclear pore complex (NPC) 1 is effected by specific targeting signals called nuclear localization sequences (NLSs) (1, 2), and is a receptor-mediated (3, 4), energy-dependent (5, 6) process. The key factors involved are members of the NLS-recognizing importin/karyopherin family (7-11), the monomeric GTPase Ran/TC4 (12, 13), and auxiliary proteins such as NTF2/p10 (14, 15). In the first step, the NLS-containing protein is recognized by the importin (IMP) heterodimer through the NLS-binding IMP␣ subunit (3, 7, 9) and targeted to the NPC through the affinity of the IMP␤ subunit (8, 10, 11, 16) for NPC components (17, 18). In the second step requiring cytoplasmic RanGDP (19,20), the transport complex is translocated through the NPC (21), and IMP␣ and the NLS-bearing protein are released into the nucleoplasm through the action of Ran GTP (19). Alternative signal-mediated nuclear import pathways have recently been identified, where either IMP␤ itself (22-24) or related homologs (25-27) fulfill the role of both IMP␣ and -␤ in binding NLSs and targeting them to the NPC (25,26,28).Although NLS receptors from different species share structural and functional homology, experimental evidence suggests that nuclear import in plant cells has unique features compared with that in other eukaryotes. In contrast to the latter, in vitro transport in plant cells appears not to be inhibited by the nucleoporin-binding lectin wheat germ agglutinin and to occur at low temperature and in the absence of exogenously added cytosol (see Refs. 29 and 30). In addition, the NLS-binding IMP␣ subunit from Arabidopsis thaliana (At-IMP␣) shows nuclear envelope association (30,31) in similar fashion to IMP␤ in mammalian and other cell systems (8, 10, 11); IMP␣ in mammalian and yeast systems shows predominantly nucleoplasmic location as well as cell cycle-dependent localization in either cytoplasm or nucleus in Drosophila (32). A linkage of At-IMP␣ with the cytoskeleton has also recently been demonstrated, with a mechanistic role in nuclear import surmised (33). Because of these novel properties, we set out to quantitate the NLS binding properties of At-IMP␣ for the first time using an ELISA-based assay (34, 35). We find that At-IMP␣ binds NLSs of different types with high affinity independent of an IMP␤ subunit, in contrast to the IMP␣ subunits from mouse and yeast, which require their respective IMP␤ subunits to achieve high affinity binding (21, 34 -36). At-IMP␣, together with Ran/ TC4 and NTF2 and in the absence of IMP␤, was able to mediate nuclear import in vitro to levels comparable with those mediated by mouse IMP␣/␤ (m-IMP␣/␤). m-IMP␣ was unable to mediate nuclear import in the absence of m-IMP␤. At-IMP␣ thus shows unique properties, being able to fulfill both NLS recognition and nuclear import in the absence of IMP␤.

Targeted intracellular delivery of photosensitizers to enhance photodynamic efficiency

“…All classes of NLS are held to be recognized specifically by the 'NLS-receptor', the α/β-importin heterodimer, during the first step of nuclear transport, as has been shown directly for the importins from several species. [103][104][105][106][107][108] Certain apparently conventional NLS have been demonstrated to be recognized specifically not by the α/β-importin heterodimer, but by importin β (importin β1) alone, such as the T cell protein tyrosine phosphatase, 109 the HIV-1 Rev protein, 110 the yeast transcriptional activator GAL4 111 and parathyroid hormone related protein (PTHrP). 112 All of these proteins appear to be able to be transported to the nucleus in the absence of importin α, which has been shown directly for PTHrP.…”

HASTY, theArabidopsisortholog of exportin 5/MSN5, regulates phase change and morphogenesis

“…Furthermore, plants possess homologs of many of the proteins known to be involved in nucleocytoplasmic transport in other organisms. These include homologs of importin α (Ballas and Citovsky, 1997;Hubner et al, 1999;Jiang et al, 1998a;Jiang et al, 2001;Smith et al, 1997a;Smith et al, 1997b), importin β (Jiang et al, 1998a), the exportin Crm1/Xpo1 (Haasen et al, 1999), Ran1 (Haizel et al, 1997), RanGAP (Rose and Meier, 2001) and RanBP1 (Kim et al, 2001). Plant homologs of importin α (Hubner et al, 1999;Jiang et al, 1998a;Jiang et al, 2001) and importin β (Jiang et al, 1998b) have also been shown to mediate nuclear transport in animal cell systems, although with somewhat different properties than the homologous yeast and mammalian proteins.…”

Crystal Structure of Rice Importin-α and Structural Basis of Its Interaction with Plant-Specific Nuclear Localization Signals

“…Some further unique features of the classical nuclear import pathway in plants have also been noted. In Arabidopsis thaliana, Impa has been reported to mediate nuclear transport independent of Impb (Hübner et al, 1999). Impa proteins from Arabidopsis and rice (Oryza sativa) display broader specificity than their mammalian counterparts (Hicks and Raikhel, 1995;Smith et al, 1997;Jiang et al, 1998;Tzfira et al, 2000).…”