Biophysical Characterization of Interactions Involving Importin-α during Nuclear Import

Abstract

Proteins containing the classical nuclear localization sequences (NLSs) are imported into the nucleus by the importin-␣/␤ heterodimer. Importin-␣ contains the NLS binding site, whereas importin-␤ mediates the translocation through the nuclear pore. We characterized the interactions involving importin-␣ during nuclear import using a combination of biophysical techniques (biosensor, crystallography, sedimentation equilibrium, electrophoresis, and circular dichroism). Importin-␣ is shown to exist in a monomeric autoinhibited state (as- Nucleocytoplasmic transport occurs through nuclear pore complexes, large supramolecular structures that penetrate the double lipid layer of the nuclear envelope. Most macromolecules require an active, signal-mediated transport process that enables the passage of particles up to 25 nm in diameter (ϳ25 MDa). The first and best characterized nuclear targeting signals are the classical nuclear localization sequences (NLSs) 1 that contain one or more clusters of basic amino acids (1). NLSs do not conform to a specific consensus sequence and fall into two distinct classes termed monopartite NLSs, containing a single cluster of basic amino acids, and bipartite NLSs, comprising two basic clusters.Despite the sequence variability, the classical NLSs are recognized by the same receptor protein termed importin or karyopherin, a heterodimer of ␣ and ␤ subunits (for recent reviews see Refs. 2-6). Importin-␣ contains the NLS-binding site, and importin-␤ is responsible for the translocation of the importincargo complex through the pore. Transfer through the pore is facilitated by the proteins Ran (Ras-related nuclear protein) and nuclear transport factor-2. Once inside the nucleus, importin-␤ binds to Ran-GTP to effect the dissociation of the import complex; the importin subunits return to the cytoplasm separately and without the cargo. The directionality of nuclear import is thought to be conferred by an asymmetric distribution of the GTP-and GDP-bound forms of Ran between the cytoplasm and the nucleus. This distribution is in turn controlled by various Ran-binding regulatory proteins.Importin-␣ consists of two structural and functional domains, a short basic N-terminal importin-␤-binding (IBB) domain (7-9), and a large NLS-binding domain comprising armadillo (Arm) repeats (10). The monopartite NLSs bind at a major site located between the first and fourth Arm repeats and additionally at a minor site spanning repeats 4 -8 (11-13). The bipartite NLSs span the two binding sites, with each site recognizing one of the basic clusters (12, 13). The linker sequence between the two basic clusters makes few contacts with the receptor, consistent with its tolerance to mutations. The affinity of the importin-targeting sequence interaction is a critical parameter in determining transport efficiency (3).The structure of mouse importin-␣ showed that in the absence of importin-␤ or NLS-containing proteins, a part of the IBB domain occupies the major NLS-binding site (14). Based on this observation, it was sugges...

The Auto-inhibitory Function of Importin α Is Essentialin Vivo

“…Generation of Mutant Importin ␣ Proteins-Previous structural and in vitro analyses identified an auto-inhibitory sequence within the N-terminal IBB domain of importin ␣ (23,24,26). The structural studies suggested that this auto-inhibitory domain interacts with the NLS binding pocket by mimicking an NLS sequence (23,24).…”

“…In support of this hypothesis, in vitro binding studies have shown that importin ␣ lacking the proposed auto-inhibitory domain (⌬IBB-␣), binds more tightly to NLS-cargo than full-length importin ␣ (26), and that this auto-inhibition of full-length importin ␣ binding to NLS-cargo is relieved in the presence of importin ␤ (17,26). Further analysis of the N-terminal IBB domain of importin ␣ revealed a proposed internal NLS that could serve as an autoinhibitory sequence to regulate NLS binding through intramolecular competition for the NLS binding site (23,24). Taken together, these studies suggest a dual role for the N-terminal domain of importin ␣ in 1) binding to importin ␤ and 2) autoinhibition of NLS-cargo binding.…”

“…The convergence of data from structural analyses and in vitro binding studies provides insight into how importin ␣ binds to and regulates interactions with NLS-cargo (17,(23)(24)(25)(26). Domain analysis has shown that importin ␣ has an N-terminal domain that binds importin ␤ (the importin ␤ binding domain or IBB), a central armadillo domain that constitutes the NLS binding pocket, and a C-terminal region that appears to be important for binding to its export receptor Cse1p/CAS (21,27).…”

Computationally Designed Armadillo Repeat Proteins for Modular Peptide Recognition

“…The strongest affinities of natural ArmRPs were reported to be in the range of 20 nM for importin-α binding to NLS peptides [27,28]. From an initial version of a consensus ArmRP library, a binder to neurotensin was selected with a K d of 7 µM [29], but the binding mode was different from the intended canonical binding [30].…”

Visualizing single molecules interacting with nuclear pore complexes by narrow-field epifluorescence microscopy

“…Fluorescent cargo concentrations were ≥ 100 nM in ensemble (bulk) experiments, and ∼100 pM for single molecule transport assays. At these cargo and cofactor concentrations, >99% of the cargo was expected to be complexed with importin-α and importin-β [33].…”