Eukaryotic cells have nuclear envelope that creates two distinct cellular compartments in
the cells. Even though these two separated compartments have unique functions by themselves,
concert, communication between the nucleus and the cytoplasm by exchange of proteins and
other molecules are needed. The nuclear import refers to the process by which specific molecules
move into the nucleus, and the nuclear export refers to the process by which specific molecules
come out from the nucleus. The nuclear pore complexes (NPC), which is a large protein complex
that crosses the nuclear envelope, is the place that allows the exchange components to get in/out
of the nucleus (Alber, Dokudovskaya et al. 2007). Nucleoporins are the major proteins that make
up the nuclear pore complex (NPC). About half of the nucleoporins have a typical alpha solenoid
or beta-propeller fold structure. The other half is a group of very flexible proteins due to its
native unfolded characteristic. The components of the NPC allow the pore to be dilated to around
26 nanometers wide from 9 nanometers at the opening status.
1.4.1 Nuclear localization signal (NLS)
NLS is a short stretch of amino acids that mediates the transportation of proteins into the
nucleus. Typically, NLS consists of one or more short sequences of positively charged lysines or
arginines. Some NLSs are glycine-rich with few positive charged residues (Bonifaci, Moroianu
et al. 1997). The NLSs can be divided into classical NLSs and non-classical NLSs. Classical
NLSs can be divided into two types: monopartite and bipartite (Boulikas 1994). Typically, the
monopartite motif has a cluster of basic residues preceded by a helix-breaking residue. The
bipartite motif has two clusters of basic residues with 9-12 non-charged residues in between.
Classical NLSs can be recognized by importin-α. Interestingly, most of the non-classical NLSs
can be recognized by importin-β (considered as import mediate). Studies show that quite a lot of
1.4.2 Nuclear exportation signal (NES)
NESs have a very important function in regulating the subcellular location of proteins.
Subcellular translocation of proteins affects the transcription and other nuclear processes. The
well known NESs are leucine-rich sequence motifs. The first NESs were identified in 1995 in the
human immunodeficiency virus type 1(HIV-1) Rev protein (Fischer, Huber et al. 1995) and
cAMP-dependent protein kinase inhibitor (PKI) (Wen, Meinkoth et al. 1995). NESs are now
extended to the motifs that contains multiple hydrophobic amino acids residues including L, V, I,
and M. CRM1 has been illustrated to be the export receptor for proteins containing the leucine
rich NESs. The transport mediated by CRM1 can be effectively inhibited by the fungicide
leptomycin B (LMB) (Fornerod, Ohno et al. 1997), as LMB can covalently bind to a cysteine
residue in CRM1 (Kudo, Matsumori et al. 1999). RanGTP and proteins with leucine-rich NESs
are considered to bind cooperatively to CRM1 and form a ternary CRM1-RanGTP-NES complex
(la Cour, Kiemer et al. 2004). This complex can be diffused to the cytoplasm, where GTP is
hydrolysed and the NES-cargo protein is released. CRM1-RanGDP will diffuse back to the
nucleus and be reused. CRM1 mediated export is a tightly regulated process, as not all NES
substrates can be exported constitutively from the nucleus. The NES-dependent export is
regulated by several mechanisms: masking/unmasking of NESs (Craig, Zhang et al. 2002),
phosphorylation (Brunet, Kanai et al. 2002), and forming a disulfide bond (Kuge, Arita et al.
2001).
1.4.3 The process of nucleocytoplasmic shuttling
Nucleocytoplasmic shuttling refers to the transport of water-soluble molecules across the
nuclear envelope by a back and forth manner. The transported water-soluble molecules include
a one-way manner, such as RNA moves from the nucleus to the cytoplasm (Rodriguez,
Dargemont et al. 2004). Others can be transported out and into the nucleus. Many proteins
(Rehberg, Lischka et al. 2002; Lahaye, Lespinasse et al. 2010) have been reported to shuttling
between the nucleus and the cytoplasm. Although small molecules (<30 kDa) cross the NPC by
simple diffusion, large molecules may need to carry a specific signal sequence (NLS and/or NES)
with the help of the nucleoporins to get through the nuclear envelope.
Ras-related nuclear protein (Ran) is a small 25 kDa protein that is known to be
transported into and out of the cell nucleus during interphase (Moore and Blobel 1994; Dasso
and Pu 1998). Ran is a GTP binding protein. Ran exists in the cell in GDP-bound and GTP-
bound forms. With the action of RCC1 (RanGEF, Ran Guanine nucleotide Exchange Factor),
RanGDP can be converted to RanGTP. The GTPase activity of Ran can be activated by
interaction with Ran GTPase activating protein (RanGAP). The conversion of RanGTP to
RanGDP is through the activation of the Ran GTPase. Since RCC1 is located inside of the
nucleus and RanGAP is bound to the cytoplasmic side of the nuclear pore complex, the ratio of
RanGTP to RanGDP is high inside the nucleus and low outside the nucleus. Meanwhile, the
protein concentration of Ran is higher in the nucleus than that in the cytoplasm. The nucleo-
cytoplasmic gradient of RanGTP powers the import cycle. Ran interacts with karyopherins (act
as importin or exportin) and changes their ability to bind or release cargo molecules, therefore
facilitates the transport of proteins across the NPC. When the cargo proteins carry NLS(s), the
proteins bind to importin and are transported into the nucleus. In the nucleus, RanGTP binds to
the importin and the imported cargo protein are released. Importin will then be diffused back to
bind to the exportin together with RanGTP. When GTP is hydrolyzed to GDP outside of the
nucleus, the exported cargos are released.
1.4.4 The function of nucleocytoplasmic shuttling of proteins
After the demonstration of nucleolin as the first nucleocytoplasmic shuttling protein in
1989 (Borer, Lehner et al. 1989), many different nucleocytoplasmic shuttling proteins have been
identified. They are many RNA binding proteins (Nakielny and Dreyfuss 1999; Shyu and
Wilkinson 2000), cell cycle regulators (Yang and Kornbluth 1999), transcription factors
(Cartwright and Helin 2000), transport receptors and adaptors (Gorlich and Kutay 1999;
Nakielny and Dreyfuss 1999), and steroid hormone receptors (Hache, Tse et al. 1999). The
nucleocytoplasmic shuttling proteins can translocate between the cytoplasm and the nucleus.
Therefore, shuttling proteins can be factors for conveying information between nucleus and
cytoplasm in the cells. The nucleocytoplasmic shuttling proteins have multiple functions
depending on the nature of the proteins. Studies have shown that nucleocytoplasmic shuttling
protein have a function in signal transduction because of their cellular location (Gama-Carvalho
and Carmo-Fonseca 2001). The translocation of proteins can regulate the cell cycle progression
and proliferation (Kau and Silver 2003). Shuttling proteins may also have a function in coupling
nuclear and cytoplasmic mRNA metabolism. Misregulation of the subcellular location was
shown to be involved in cancers (Takai, Tan et al. 2005).