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Page 142 Sendino et al. Cancer Drug Resist 2018;1:139-63 I http://dx.doi.org/10.20517/cdr.2018.09

In fact, it has been shown that, by artificially raising the concentration of RanGTP in the cytoplasm, the
[25]
direction of the transport can be inverted .

Beyond the basic transport machinery described above, multiple additional mechanisms may contribute
to regulate the nucleocytoplasmic distribution of a given protein in a dynamic and finely-tuned manner.
These mechanisms include post-translational modifications, such as phosphorylation [26,27] or ubiquitination
[28]
(reviewed by Rodríguez ), as well as masking/unmasking of the transport signals by homo/heterodimer-
ization [29,30] .

XPO1-mediated protein nuclear export: cargos, mechanisms and signals
XPO1 has a wide repertoire of cargos, including not only cellular proteins, but also viral proteins expressed
[31]
in infected cells (reviewed by Ding et al. ).
Identification of XPO1 cargos has been greatly facilitated by the use of LMB as an inhibitor. In cellular
experiments, cytoplasmic XPO1 cargos often relocate to the nucleus in the presence of LMB. This experi-
mental approach cannot be used to demonstrate XPO1-mediated export of proteins that are constitutively
located to the nucleus. An alternative approach in this case could be ectopic overexpression of the receptor,
which promotes export of NES-containing nuclear cargos to the cytoplasm . Besides LMB-based experi-
[32]
ments, the identification, validation and characterization of XPO1 cargos often involve biochemical analy-
ses to demonstrate RanGTP-dependent binding, as well as mutagenesis to map the NES. In over 15 years of
research, hundreds of individual proteins were studied using these approaches and around 200 bona-fide
XPO1-exported cargos were identified . More recently, the introduction of tandem mass spectrometry
[33]
(MS/MS)-based high throughput analyses has expanded the repertoire of potential XPO1 cargos (the so-
[34]
called “XPO1 exportome”) to above 1000 cellular proteins , although many of them still need to be fur-
ther validated and their NESs identified.

The search for novel cargos is still on-going, and continues to provide further insight into the physiological
relevance of XPO1. For example, it has been recently found that the NES-containing protein POST and the
ubiquitin-binding protein UBIN form a complex that mediates XPO1-dependent nuclear export of polyu-
[35]
biquitinated proteins , a process that seems to be exacerbated in cancer cells treated with the proteasome
[36]
inhibitor bortezomib . These findings reveal a novel role for XPO1 in nuclear protein homeostasis that
might also have important implications for cancer therapy.
From a mechanistic point of view, XPO1-mediated nuclear export consists essentially in the binding of an
NES-containing protein in the nucleus and its release in the cytoplasm. The XPO1/NES interaction has low
affinity, and needs to be stabilized by the cooperative binding of nuclear RanGTP, facilitated by the cofac-
tor RanBP3 [37-39] . Structural and biochemical studies carried out over the last decade have contributed to
dissecting the series of molecular events that underlie the cycle of assembly and disassembly of the XPO1/
[13]
[12]
[11]
RanGTP/NES complex (reviewed by Koyama and Matsuura , Fung and Chook and Monecke et al. ).
As schematically illustrated in Figure 2A, XPO1 is a ring-shaped protein with a concave inner surface and
a convex outer surface. RanGTP binds to the inner surface, and NESs dock into a hydrophobic groove in
the outer surface of the receptor. The open/close state of the NES binding groove is allosterically regulated
by conformational rearrangements of two additional XPO1 structural elements, termed the H9 loop and
the C-terminal extension, which play a crucial role in the cycle of NES binding and release.

As illustrated in Figure 1C, “leucine-rich” NESs conform to a loose consensus sequence with a characteris-
tic spacing of hydrophobic residues [40,41] . Hundreds of different amino acid sequences have been experimen-
tally validated as bona-fide NESs that bind the receptor with different affinity, and may be exported with
different efficiency [33,42,43] . This high variability can be explained by the recent finding that NESs with differ-