Macromolecules require nuclear import receptors for transport across the nuclear membrane pore (Conti et al., 2006
; Pemberton and Paschal, 2005
). The Karyopherins Importin-α and -β mediate a well-studied nuclear import system in eukaryotic cells (Catimel et al., 2001
; Cingolani et al., 1999
; Conti et al., 1998
). Imp-β interacts with nuclear localization signals in either cargo proteins or the adaptor Imp-α which in turn binds cargo (Goldfarb et al., 2004
). Imp-β interacts with nucleoporins at the nuclear pore complex to enable the selective transport of macromolecules across the nuclear membrane (Cook et al., 2007
). The adaptor protein Imp-α exhibits a two-domain architecture: an N-terminal Imp-β binding (IBB) domain connected by a linker to an ARM-repeat C-terminal domain. The IBB includes basic-residue rich regions that resemble a bipartite NLS that is recognized by Imp-β (Dingwall and Laskey, 1991
)(Cingolani et al., 1999
). To regulate protein shuttling, the concave side of the Imp-α C-terminal domain can recognize either the cargo protein or its own IBB in an auto-inhibitory fashion (Conti et al., 1998
). This auto-inhibition explains the Imp-α switch between the cytosolic high-affinity for cargo and the nuclear low-affinity that results in directional transport (Fanara et al., 2000
; Kobe, 1999
; Kobe and Kemp, 1999
Here, we report that like Imp-α Nro1 has a two-domain architecture with a flexible NTD and all α-helical repeat CTD. Functional studies reveal that Nro1 defines a new class of helical repeat, nuclear import adaptor. Several lines of evidence support this conclusion. First, Nro1 is required for nuclear localization of Ofd1 (). Second, Nro1 binds the Ofd1 cargo molecule through its NTD, not its C-terminal helical-repeat domain like Imp-α (). Third, the Nro1 NTD does not possess the auto-inhibitory property of the Imp-α IBB domain, since removal of the Nro1 NTD α0
(Δ10–29) blocks, rather than increases, cargo binding ()(Kobe, 1999
). Fourth, both Nro1 and Ofd1 require the Imp-β family member Kap123 for nuclear import (), suggesting that Nro1 couples Ofd1 to a Karyopherin β for transport.
Nro1 functions as an adaptor and is structurally similar to both Imp-α, an ARM-repeat adaptor that imports NLS-containing cargo to the nucleus (Conti et al., 1998
), and VPS35, a HEAT-repeat adaptor in the retromer cargo-recognition complex that sorts acid hydrolases to the lysosome (Hierro et al., 2007
). These two proteins use their α-helical-repeat domains to recognize cargo, although in different manners. Imp-α recognizes only the cargo NLS peptide in a cleft on the concave side of its C-terminal domain (Catimel et al., 2001
). VPS35 binds by wrapping itself around the cargo protein with most of the concave surface participating in the recognition (Hierro et al., 2007
). Unlike these adaptor proteins, the Nro1 CTD is relatively less important for recognition of Ofd1 ( and ).
In contrast, our data indicate a major role for the Nro1 NTD helix α0 in binding Ofd1 and a less important role for the concave surface of the Nro1 CTD (, and ). In vitro experiments using Nro1, Nro1(Δ1–30), and the C-terminal degradation domain of Ofd1 indicate a direct interaction between the Ofd1CTDD and the first thirty residues of Nro1. Thermodynamic data obtained from Nro1-Ofd1CTDD complex formation showed an unfavourable entropic contribution to the binding (ΔS = −13 kcal mol−1 K−1), consistent with the notion that complex formation induces order in an otherwise disordered domain like the NTD. The removal of the Nro1 regions that include α0 abrogates Nro1 binding to Ofd1CTDD. Furthermore, we show by yeast two-hybrid that the NTD interacts with Ofd1CTDD, and GFP-NTD is sufficient to inhibit the effect of endogenous Ofd1 in Sre1N degradation (). Compared to large deletions in the NTD, select amino acid substitutions in the concave side of the CTD () showed relatively minor negative effects on binding and nuclear localization of Ofd1 ( and ). These mild effects may be explained by the small footprint of the selected substitutions within what could be a large area of recognition if Nro1 employs a VPS35-like mode of recognition.
Nro1 regulates the fission yeast hypoxic response by inhibiting Ofd1 function in Sre1N degradation. Nro1’s newly discovered role as a nuclear import adaptor raised the question of whether Nro1 simply regulates Sre1N by controlling the localization of Ofd1. For example, in nro1Δ cells Sre1N may be rapidly degraded due to the separation of Ofd1 from a hypothetical inhibitor localized in the nucleus. However, fusing a nuclear localization sequence to Ofd1 restored its nuclear localization, but had no impact on Sre1N stability (). These results indicate that the rapid degradation of Sre1N is not a consequence of Ofd1 localization, but rather due to the ability of Nro1 to bind and directly inhibit Ofd1. Thus, we propose that Nro1 has two different functions that require binding to Ofd1: (1) Nro1 is required for Ofd1 nuclear localization, and (2) Nro1 binding is required to inhibit Ofd1-mediated degradation of Sre1N.
Taken together these results suggest a mechanism for the Nro1-dependent regulation of Sre1N (). In the cytosol, Nro1 and Ofd1 form a heterodimer that is shuttled into the nucleus by a pathway that requires Kap123. Under low oxygen, the Nro1-Ofd1 heterodimer is more stable, which prevents Ofd1CTDD
interaction with Sre1N (Lee et al., 2009
). Under normoxic conditions, the interaction between Nro1 and Ofd1 is disrupted. Ofd1 is free to interact with Sre1N accelerating its turnover by the proteasome. One hypothesis for the oxygen-dependence of this mechanism is that in the presence of oxygen the enzymatic activity of the Ofd1 dioxygenase domain may modify Ofd1CTDD
, Nro1, or both thereby interfering with the Nro1-Ofd1 heterodimer stability. The Ofd1 dioxygenase domain is presumably also active in the cytosol. However, nuclear localization of Ofd1 is not affected by inhibition of the Ofd1 dioxygenase domain (data not shown). Perhaps Kap123 further stabilizes the Nro1-Ofd1 complex, thus allowing nuclear import in the presence of oxygen ().
Model for mechanism of Sre1N regulation by Nro1-Ofd1
The work presented here shows that Nro1 defines a new class of nuclear import adaptor that is required for the nuclear transport of Ofd1. Understanding how Nro1 itself enters the nucleus, the role of Kap123 in this process, and what other cargo require Nro1 are important questions for future studies. Nro1 has a second function as a direct inhibitor of Ofd1CTDD in the regulation of hypoxic gene expression mediated by Sre1. Regulation of this hypoxic response triggered by oxygen is completely dependent on Nro1-Ofd1 complex dynamics. Elucidation of the molecular details of this interaction is the focus of current studies.