Structural Basis for the Specificity of Bipartite Nuclear Localization Sequence Binding by Importin-α

Abstract

Importin-␣ is the nuclear import receptor that recognizes cargo proteins carrying conventional basic monopartite and bipartite nuclear localization sequences (NLSs) and facilitates their transport into the nucleus. Bipartite NLSs contain two clusters of basic residues, connected by linkers of variable lengths. To determine the structural basis of the recognition of diverse bipartite NLSs by mammalian importin-␣, we co-crystallized a non-autoinhibited mouse receptor protein with peptides corresponding to the NLSs from human retinoblastoma protein and Xenopus laevis phosphoprotein N1N2, containing diverse sequences and lengths of the linker. We show that the basic clusters interact analogously in both NLSs, but the linker sequences adopt different conformations, whereas both make specific contacts with the receptor. The available data allow us to draw general conclusions about the specificity of NLS binding by importin-␣ and facilitate an improved definition of the consensus sequence of a conventional basic/bipartite NLS (KRX 10 -12 KRRK) that can be used to identify novel nuclear proteins.Nucleocytoplasmic transport occurs through nuclear pore complexes, large proteinaceous 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 best characterized nuclear targeting signals are the conventional nuclear localization sequences (NLSs) 1 that contain one or more clusters of basic amino acids (1). The NLSs fall into two distinct classes termed monopartite NLSs, containing a single cluster of basic amino acids, and bipartite NLSs, comprising two basic clusters separated by a spacer. Despite the variability, the conventional basic NLSs are recognized by the same receptor protein termed importin or karyopherin, a heterodimer of ␣ and ␤ subunits (for recent reviews, see Refs. 2-4). Importin-␣ (Imp␣) contains the NLSbinding site, and importin-␤ (Imp␤) is responsible for the translocation of the importin-substrate complex through the nuclear pore complex. Once inside the nucleus, Ran-GTP binds to Imp␤ and causes the dissociation of the import complex. Imp␣ becomes autoinhibited, and both importin subunits return to the cytoplasm separately without the import cargo. The directionality of nuclear import is 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.Imp␣ consists of two structural and functional domains, a short basic N-terminal Imp␤-binding domain (5-7) and a large NLS-binding domain built of armadillo (Arm) repeats (8). The structural basis of monopartite and bipartite NLS recognition by Imp␣ has been studied crystallographically in yeast and mouse Imp␣ proteins (9 -11). The two basic clusters of the bipartite NLSs bind to two separate binding sites on Imp␣, involving Arm repeats 1-4 and 4 -8, respectivel...

The Bipartite Nuclear Localization Sequence of Rpn2 Is Required for Nuclear Import of Proteasomal Base Complexes via Karyopherin αβ and Proteasome Functions

“…3A). Further basic amino acid residues were found within the C-terminal region of each ATPase subunit, which hardly conformed with classic NLS (25). In this work, we focused on NLS of base subunits, especially of domains conserved from yeast to human.…”

“…Both peptides efficiently targeted GFP into the nucleus and were specifically recognized by karyopherin ␣␤. The Rpn2 NLS fulfills the structural requirements of a bipartite NLS, whereas the Rpt2 NLS consists of two monopartite NLS spaced by 17 amino acid residues, which exceed the common linker length of a bipartite NLS (25).…”

Parafibromin is a nuclear protein with a functional monopartite nuclear localization signal

“…Nuclear proteins need to be imported from the cytoplasm to the nucleus, and this translocation across the nuclear envelope is facilitated by a soluble heterodimeric receptor comprising importin a and importin b (Hodel et al, 2001;Fontes et al, 2003). This process depends on the recognition, by importins a and b1, of clusters of basic amino acids, which are referred to as nuclear localization signals (NLSs), in the protein (Nakielny and Dreyfuss, 1999;Quadrini and Bieker, 2002).…”

Nuclear localization signals and human disease

“…Co-crystal structures of both S. cerevisiae and M. musculus importin-a have been solved in complex with various NLS peptides (52)(53)(54)(55)(56)(57)(58)(59). The three-dimensional structures reveal that NLS peptides bind specifically in two binding grooves created by flexible armadillo (ARM) motifs in the central domain of importin-a (54, 55).…”