Cell Nucleus Biogenesis, Structure and Function
399
These states establish a RanGTP gradi-
ent across the nuclear pore, in which Ran
inside the nucleus is predominantly GTP-
bound, while the one outside is mostly in
the GDP-bound state. This gradient is ma-
nipulated by a variety of accessory factors
that together regulate transport. The most
important feature of the RanGTP/GDP
cycle is that GTP loading occurs in the
nucleus while hydrolysis occurs in the
cytoplasm. The Ran guanine nucleotide
exchange factor (RanGEF) called RCC1
catalyzes GTP loading, while RanGTP hy-
drolysis is performed by the RanGTPase
activating protein RanGAP together with
accessory proteins RanBP1 and RanBP2.
RanGAP and RanBP1/2 are restricted to
the cytoplasm. This asymmetrical distri-
bution of the Ran regulators establishes a
RanGTP gradient such that each molecule
of Ran that enters the nucleus will be
rapidly converted to the GTP-bound state,
while RanGTP moving out of the nu-
cleus will be converted to RanGDP on
entering the cytoplasm. The magnitude
of this gradient can be estimated using
Ran biosensors that use fluorescence res-
onance energy transfer (FRET) to probe
structural changes during transport. This
approach suggests that the concentration
of free RanGTP in the nucleus is at least
200-fold greater than that in the cytoplasm.
The RanGTP gradient provides the
position information that drives nuclear
transport. During import, the importins
will bind to their substrates in the absence
of Ran and after translocation release
the substrate on binding RanGTP in
the nucleus. Here, the RanGTP operates
as a kind of exchange factor and the
importin–RanGTP complex that forms is
recycled back to the cytoplasm. During
export, the exportins only bind to their
cargoes in the presence of RanGTP. The
RanGTP gradient ensures that this occurs
almost exclusively in the nucleus. On the
cytoplasmic side of the pore complex, the
exportin/RanGTP/cargo complex decays
following RanGTP hydrolysis that is driven
by RanGAP and RanBP1/2. RanGDP
returns to the nucleus in a complex
with a Ran importer protein called NTF2.
Once inside the nucleus, RanGDP is once
again converted to the GTP-bound form,
to release NTF2 so that a new cycle of
transport can begin.
3.4.2
RNA Export Pathways
In contrast to the well-characterized mech-
anisms used to regulate protein transport
through nuclear pores, many details of the
mechanisms for RNA transport remain
sketchy. The importance of this should
not be underestimated as it clearly repre-
sents a critical point of regulation in the
control of gene expression. In addition, it is
well known that many independent path-
ways regulate the transport of different
classes of RNA–protein complexes – this
could be potentially very complex allow-
ing the differential regulation of speci±c
classes of RNA transcript in response to a
variety of developmental and growth regu-
latory cues.
Basic details of the primary mechanisms
that control mRNP export have been eluci-
dated. The molecular interactions that un-
derlie this process are dependent on events
that take place at the site of mRNA syn-
thesis. The critical components are small
proteins that are part of a much larger com-
plex that is deposited on the mRNA during
splicing. The complex called the – 20/24
complex because of its location at about 20
nucleotides upstream of the exon–intron
junction contains a number of proteins,
but includes the protein Aly. Aly inter-
acts speci±cally with TAP/mex67, which
serves as a major export complex for mR-
NAs. It is important to recognize that
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