The separation of genetic material from the cytoplasm by the double membrane of the nuclear envelope (NE) is the major characteristic of all eukaryotic organisms. The inner nuclear membrane and the outer nuclear membrane of the NE are separated by the perinuclear space. The outer nuclear membrane forms a continuum with the endoplasmic reticulum (ER). The exchange of macromolecules such as proteins or RNA across the nuclear envelope depends on highly regulated import and export processes (Meier and Somers, 2011). For this, nuclear pore complexes (NPC) represent tunnels that span the nuclear envelope (Figure 1.1). NPCs have an eightfold-symmetry and are composed of numerous nucleoporin proteins that form distinct sub-complexes (Suntharalingam and Wente, 2003; Hoelz et al., 2011). Nucleoporins either belong to the central FG nucleoporins (named after hydrophobic phenylalanine-glycine (FG)-rich motifs) located in the central channel or build the cytoplasmic filaments, cytoplasmic ring, nuclear ring and nuclear basket. (Cronshaw et al., 2002; Tamura and Hara-Nishimura, 2011). Selective transport through the NPCs is regulated by the FG nucleoporins that represent docking sites for transport receptors (Hoelz et al., 2011). These proteins limit the diffusion of molecules into the nucleus by engaging in low-affinity and high-specificity interactions with transport factors (Alber et al., 2007; Ryan and Wente, 2000; Cronshaw et al., 2002;
Strawn et al., 2004). Although the general structure is highly conserved in eukaryotes, number and size of NPCs are variable between different organisms cell types and even the developmental stage of a given cell type influences NPC composition (Fiserova et al., 2009; Maul, 1977; Goldberg et al., 1997; Reichelt, 1990; Rout, 1993; Kiseleva et al., 2001;
Winey et al., 1997; Capelson and Hetzer, 2009). For Arabidopsis 30 putative nucleoporins (NUPs) that are conserved among yeast, vertebrates and plants have been identified (Tamura et al., 2010; Tamura and Hara-Nishimura, 2011; Neumann et al., 2010).
Introduction _______________________________________________________________
Due to the action of FG nucleoporins only small soluble molecules and proteins with a molecular weight of less than 40-60 kDa can translocate into the nucleus by passive diffusion (Stewart, 2007a; Wang and Brattain, 2007). Hence, an additional set of proteins is involved in transport of larger proteins across the NE: the nuclear transport receptors (NTRs) of the karyopherin family. NTRs mediate both, nuclear import and export and the respective receptors are thus categorized into importins and exportins (Meier, 2007).
Nucleocytoplasmic translocation usually requires the presence of a nuclear localization signal (NLS) for nuclear destination or leucine-rich nuclear export signal (NES) for cytoplasmic destination on the cargo substrate (Figure 1.2, Görlich and Kutay, 1999; Terry et al., 2007; Stade et al., 2002). The most abundant NLS motifs are basic Lys/Arg-rich sequences that can be monopartite with the consensus sequence (K[K/R]X]K/R]) or bipartite ([K/R][K/R]X10-12[K/R]3/5, Chang et al., 2013; Marfori et al., 2011; Marfori et al., 2012).
Asymmetrical distribution of the small GTPase Ran (Ras-related nuclear protein) between the nucleus and the cytoplasm is the driving force of nucleocytoplasmic
Figure 1.1 Schematic overview of karyopherin-mediated protein transport through a nuclear pore complex. In the cytoplasm, a trimeric complex of the two import receptor subunits IMP-α and IMP-β with an NLS containing cargo protein is formed. IMP-α directly binds to the NLS and thereby bridges the interaction of cargo to IMP-β. One of the Arabidopsis IMP-α proteins is MOS6 (MODIFIER OF SNC1 6). IMP-β mediates interaction with nucleoporins in the nuclear pore complex for translocation into the nucleoplasm. The nuclear pore complex consists of distinct sub-complexes: the cytoplasmic filaments, cytoplasmic ring, nuclear ring, nuclear basket and the core/central channel. The trimeric transport complex dissociates in the nucleus by action of Ran in its GTP-bound form. Cargo export is mediated by exportin together with Ran in its GTP-bound form along the concentration gradient for Ran-GTP. In the Cytoplasm, cargo and exportin dissociate after GTP hydrolysis.
Figure adapted from Wiermer et al. (2007).
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transport (Görlich and Kutay, 1999; Terry et al., 2007; Meier and Somers, 2011). The Ran guanine nucleotide exchange factor (RanGEF) is bound to chromatin and thus restricted to the nucleus, which leads to a higher concentration of Ran-GTP in the nucleus (Merkle, 2011). In contrast, the Ran GTPase-activating protein (RanGAP) leads to a higher concentration of Ran-GDP in the cytoplasm due to its cytoplasmic localization (Xu et al., 2007). Interestingly, many small proteins as for example many transcription factors and pathogenic effector proteins also contain NLS and rely on active nucleocytoplasmic transport to ensure efficient import, although they could freely diffuse through NPCs (Krebs et al., 2010; Ballesteros et al., 2001; Caillaud et al., 2012b; Deslandes et al., 2003;
Schornack et al., 2010; Weinthal et al., 2011).
For classical nuclear import, the NLS-containing cargo protein is bound by the receptor protein IMPORTIN-α (IMP-α) in the cytoplasm via its armadillo (ARM) repeat domains (Cook et al., 2007; Marfori et al., 2011; Chang et al., 2013). IMP-α proteins have a N-terminal auto-inhibitory IMPORTIN-β-binding (IBB) domain, ten armadillo (ARM) repeats that form two NLS-binding pockets and a C-terminal acidic patch that interacts with the CAS export protein (Conti and Kuriyan, 2000; Goldfarb et al., 2004). The Arabidopsis genome encodes nine IMP-α proteins (Wirthmueller et al., 2013). Upon cargo binding, the IBB domain of the IMP-α protein is exposed and interacts with IMPORTIN-β (IMP-β, Figure 1.2, Kobe, 1999; Harreman et al., 2003). In the nucleus, Ran-GTP binding to IMP-β causes conformational changes that result in dissociation of the trimeric complex (Gilchrist et al., 2002). The exportin CAS in its Ran-GTP bound form then interacts with IMP-α, which results in release of the cargo. IMP-α bound to CAS-Ran-GTP, as well as IMP-β-Ran-GTP are exported to the cytoplasm along the Ran-GTP gradient (Kutay et al., 1997). In the cytoplasm, Ran-GTP is hydrolyzed to Ran-GDP, which leads to the release of IMP-α and IMP-β, respectively (Stewart, 2007b). Ran-GDP is returned to the nucleus by the NUCLEAR TRANSPORT FACTOR 2 (NTF2), where it is converted back to Ran-GTP by Ran-GEF (Ribbeck et al., 1998; Bhattacharya and Steward, 2002; Zhao et al., 2006).
The Arabidopsis karyopherin EXPORTIN 1 (XPO1) mediates export of NES-motif containing cargo proteins out of the nucleus (Haasen et al., 1999; La Cour et al., 2003;
Stade et al., 1997). XPO1 interacts with Ran-GTP for nuclear export. Ran-GTP is hydrolyzed in the cytoplasm for subsequent cargo-release (Haasen et al., 1999).
Introduction _______________________________________________________________
In addition to the classical NLS presented above, atypical nuclear localization signals have been described. A number of cargo proteins with atypical NLS can directly bind to and thus be imported by IMP-β alone. Usually, these sequences are basic and structurally more complex than classical NLS (Lam et al., 1999; Lee et al., 2003; Nagoshi and Yoneda, 2001; Jakel and Görlich, 1998; Zehorai and Seger, 2014; Palmeri and Malim, 1999).
Additional non-canonical NLS are the Matα2 NLS found in yeast and maize (Hall et al., 1984; Hicks et al., 1995) and the non-canonical M9 NLS whose nuclear import is mediated by TRANSPORTIN 1 (TRN1) without involvement of IMP-α (Michael et al., 1995; Bogerd et al., 1999; Pollard et al., 1996; Lee et al., 2006). PY-NLS that contain a characteristic proline/tyrosine sequence were identified by characterization of NLSs recognized by human TRN1 (Marfori et al., 2011; Lee et al., 2006). Interestingly, Arabidopsis TRN1 is the transport receptor for two small RNA-binding proteins, AtGRP7 and AtGRP8 (GLYCINE-RICH RNA-BINDING PROTEIN 7 and 8), that are involved in plant immunity (Ziemienowicz et al., 2003).