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Signal Sequence-Based Cell-Penetrating Peptides 97
on ATP. As for small molecules, cytoplasmic retention sequences and regulatory
phosphorylation can modulate transport of larger molecules through the NPC. 14,22-24
Transport across the NPC basically involves four major steps, initiated by binding
of an NLS to importin α in the cytoplasm, which strengthens the interaction of
the latter with importin β. Importin β is responsible for the translocation of the
importin and cargo complex through the pore. This is followed by docking of the
cargo and importin complex to the cytoplasmic periphery of the NPC, then transport
through the pore, which involves hydrophobic clusters of aromatic residues (Phe)
from specific nucleoporins. 24,25 Once in the nucleus, the complex finally dissociates
in an energy-dependent mechanism, followed by recycling of the transport factors
to the cytoplasm, thus releasing them for the next import cycle. Transfer through
the pore is facilitated by a series of factors including the small GTPase protein Ran
(ras-related nuclear protein), the nuclear transport factor-2 (NTF-2), and the chromatin-bound
guanine exchange factor RCC1. The small GTPase Ran confers directionality
to the nuclear import system: Ran-GTPase allows association of the cargo
and importin complex in its GDP-form in the cytoplasmic compartment, whereas
its GTP-form dissociates the complex in the nucleus.
Once in the nucleus, importin β binds to Ran-GTP, which induces dissociation
of the import complex, and importin α is then recycled to the cytoplasm. The binding
of the cargo and importin complex to the cytoplasmic face of the NPC is ATPindependent,
whereas its nuclear accumulation is dependent on ATP. The GDP and
GTP-forms of Ran are maintained, respectively, by the Ran GTPase activity protein
1 (GAP) in the cytoplasm and the nucleotide exchange factor Ran-GEF in the
nucleoplasm. A recent study has revealed that the interaction of importin β and
importin α confers very high affinity to the complex for mono or bipartite NLS.
After translocation of the heterotrimeric complex to the nuclear compartment, Ran-
GTP induces dissociation of the importin β and importin α complex, which favors
release of the cargo in the nucleus and converts importin α into an auto-inhibited
conformation, thereby preventing it from binding to nuclear proteins. 26-28
Another major pathway of nuclear delivery has been described for the M9 NLS
sequence that mediates bidirectional transport of ribonucleoprotein hrRNP A1. M9
is a short signal sequence of 38 residues rich in glycine and aromatic residues,
encoding an NLS as well as an NES sequence. Nuclear transport is mediated by
interaction with the transport receptor protein transportin 1 (TRN1), a member of
the importin family of nucleocytoplasmic transport factors. 29 Several M9-like
sequences containing NLS–NES import and export sequences that overlap have been
identified. 29,30 A similar sequence, characterized by an NLS–NES, has been described
for Tap proteins involved in export of nuclear mRNA. Although their sequence is
not similar to that of M9, they seem to be involved in the same import mechanism
as transportin proteins. As for the β importin pathway, dissociation between transportin
1/M9 NLS is induced by Ran-GTP. 29-31
5.2.3 NLS INVOLVED IN THE DELIVERY OF ACTIVE CARGO
Synthetic NLS-containing peptides have been extensively used to improve the
nuclear delivery of cargoes (drugs, DNA). They have been attached to cargoes or