Karyopherinbeta (Kapbeta) proteins bind nuclear localization and export signals (NLSs and NESs) to mediate nucleocytoplasmic trafficking, a process regulated by Ran GTPase through its nucleotide cycle. Diversity and complexity of signals recognized by Kap betas have prevented prediction of new Kap beta substrates. The structure of Kap beta 2 (also known as Transportin) bound to one of its substrates, the NLS of hnRNP A1, that we report here explains the mechanism of substrate displacement by Ran GTPase. Further analyses reveal three rules for NLS recognition by Kap beta 2: NLSs are structurally disordered in free substrates, have overall basic character, and possess a central hydrophobic or basic motif followed by a C-terminal R/H/KX(2-5)PY consensus sequence. We demonstrate the predictive nature of these rules by identifying NLSs in seven previously known Kap beta 2 substrates and uncovering 81 new candidate substrates, confirming five experimentally. These studies define and validate a new NLS that could not be predicted by primary sequence analysis alone.

Proteins in the Karyopherinβ family mediate the majority of macromolecular transport between the nucleus and the cytoplasm. Eleven of the 19 known human Karyopherinβs and 10 of the 14 S. cerevisiae Karyopherinβs mediate nuclear import through recognition of nuclear localization signals or NLSs in their cargos. This receptor-mediated process is essential to cellular viability as proteins are translated in the cytoplasm but many have functional roles in the nucleus. Many known Karyopherinβ-cargo interactions were discovered through studies of the individual cargos rather than the karyopherins, and this information is thus widely scattered in the literature. We consolidate information about cargos that are directly recognized by import-Karyopherinβs and review common characteristics or lack thereof among cargos of different import pathways. Knowledge of Karyopherinβ-cargo interactions is also critical for the development of nuclear import inhibitors and the understanding of their mechanisms of inhibition.

CRM1 (also known as XPO1 and exportin 1) mediates nuclear export of hundreds of proteins through the recognition of the leucine-rich nuclear export signal (LR-NES). Here we present the 2.9Å structure of CRM1 bound to snurportin 1 (SNUPN). Snurportin 1 binds CRM1 in a bipartite manner by means of an amino-terminal LR-NES and its nucleotide-binding domain. The LR-NES is a combined α-helical-extended structure that occupies a hydrophobic groove between two CRM1 outer helices. The LR-NES interface explains the consensus hydrophobic pattern, preference for intervening electronegative residues and inhibition by leptomycin B. The second nuclear export signal epitope is a basic surface on the snurportin 1 nucleotide-binding domain, which binds an acidic patch on CRM1 adjacent to the LR-NES site. Multipartite recognition of individually weak nuclear export signal epitopes may be common to CRM1 substrates, enhancing CRM1 binding beyond the generally low affinity LR-NES. Similar energetic construction is also used in multipartite nuclear localization signals to provide broad substrate specificity and rapid evolution in nuclear transport.

Proline–tyrosine nuclear localization signals (PY-NLSs) are recognized and transported into the nucleus by human Karyopherin (Kap) β2/Transportin and yeast Kap104p. Multipartite PY-NLSs are highly diverse in sequence and structure, share a common C-terminal R/H/KX2–5PY motif, and can be subdivided into hydrophobic and basic subclasses based on loose N-terminal sequence motifs. PY-NLS variability is consistent with weak consensus motifs, but such diversity potentially renders comprehensive genome-scale searches intractable. Here, we use yeast Kap104p as a model system to understand the energetic organization of this NLS. First, we show that Kap104p substrates contain PY-NLSs, demonstrating their generality across eukaryotes. Previously reported Kapβ2–NLS structures explain Kap104p specificity for the basic PY-NLS. More importantly, thermodynamic analyses revealed physical properties that govern PY-NLS binding affinity: (1) PY-NLSs contain three energetically significant linear epitopes, (2) each epitope accommodates substantial sequence diversity, within defined limits, (3) the epitopes are energetically quasi-independent, and (4) a given linear epitope can contribute differently to total binding energy in different PY-NLSs, amplifying signal diversity through combinatorial mixing of energetically weak and strong motifs. The modular organization of the PY-NLS coupled with its combinatorial energetics lays a path to decode this diverse and evolvable signal for future comprehensive genome-scale identification of nuclear import substrates.

A previous bioinformatics study identified a putative PY-NLS in the yeast transcription factor Tfg2p (Suel, K. E., Gu, H., and Chook, Y. M. (2008) PLoS Biol. 6, e137). In this study, we validate Tfg2p as a Kap104p substrate and examine the energetic organization of its PY-NLS. The Tfg2p PY-NLS can target a heterologous protein into the cell nucleus through interactions with Kap104p. Surprisingly, full-length Tfg2p is still localized to the nucleus of Kap104p temperature-sensitive cells and, similarly, Tfg2p with a mutated PY-NLS is nuclear in wild-type cells. Other Karyopherin␤s (Kap␤s) such as Kap108p and Kap120p also bind Tfg2p and may import it into the nucleus. More importantly, we demonstrate that Tfg2p is retained in the nucleus through DNA binding. Mutations of DNA binding residues relieve nuclear retention and unmask the role of Kap104p in Tfg2p nuclear import. More generally, steadystate localization of a nuclear protein is dictated by its nuclear import and export activities as well as its interactions in the nucleus and the cytoplasm.