Axoplasmic proteins containing nuclear localization signals (NLS) signal retrogradely by an unknown mechanism in injured nerve. Here we demonstrate that the importin/karyopherin alpha and beta families underlie this process. We show that importins are found in axons at significant distances from the cell body and that importin beta protein is increased after nerve lesion by local translation of axonal mRNA. This leads to formation of a high-affinity NLS binding complex that traffics retrogradely with the motor protein dynein. Trituration of synthetic NLS peptide at the injury site of axotomized dorsal root ganglion (DRG) neurons delays their regenerative outgrowth, and NLS introduction to sciatic nerve concomitantly with a crush injury suppresses the conditioning lesion induced transition from arborizing to elongating growth in L4/L5 DRG neurons. These data suggest a model whereby lesion-induced upregulation of axonal importin beta may enable retrograde transport of signals that modulate the regeneration of injured neurons.

Importin ␣ plays a pivotal role in the classical nuclear protein import pathway. Importin ␣ shuttles between nucleus and cytoplasm, binds nuclear localization signal-bearing proteins, and functions as an adapter to access the importin ␤-dependent import pathway. In contrast to what is found for importin ␤, several isoforms of importin ␣, which can be grouped into three subfamilies, exist in higher eucaryotes. We describe here a novel member of the human family, importin ␣7. To analyze specific functions of the distinct importin ␣ proteins, we recombinantly expressed and purified five human importin ␣'s along with importin ␣ from Xenopus and Saccharomyces cerevisiae. Binding affinity studies showed that all importin ␣ proteins from humans or Xenopus bind their import receptor (importin ␤) and their export receptor (CAS) with only marginal differences. Using an in vitro import assay based on permeabilized HeLa cells, we compared the import substrate specificities of the various importin ␣ proteins. When the substrates were tested singly, only the import of RCC1 showed a strong preference for one family member, importin ␣3, whereas most of the other substrates were imported by all importin ␣ proteins with similar efficiencies. However, strikingly different substrate preferences of the various importin ␣ proteins were revealed when two substrates were offered simultaneously.

NF-B transcription factors are retained in the cytoplasm in an inactive form until they are activated and rapidly imported into the nucleus. We identified importin ␣3 and importin ␣4 as the main importin ␣ isoforms mediating TNF-␣-stimulated NF-B p50/p65 heterodimer translocation into the nucleus. Importin ␣3 and ␣4 are close relatives in the human importin ␣ family. We show that importin ␣3 isoform also mediates nuclear import of NF-B p50 homodimer in nonstimulated cells. Importin ␣3 is shown to directly bind to previously characterized nuclear localization signals (NLSs) of NF-B p50 and p65 proteins. Importin ␣ molecules are known to have armadillo repeats that constitute the N-terminal and C-terminal NLS binding sites. We demonstrate by site-directed mutagenesis that NF-B p50 binds to the N-terminal and p65 to the Cterminal NLS binding site of importin ␣3. In vitro competition experiments and analysis of cellular NF-B suggest that NF-B binds to importin ␣ only when it is free of IB␣. The present study demonstrates that the nuclear import of NF-B is a highly regulated process mediated by a subset of importin ␣ molecules.