Discuss the molecular mechanisms that regulate specific and directional protein

transport across the nuclear envelope.

Entry to the nucleus must be regulated; there are many proteins in the cytosol that, upon
entry to the nucleus, would be fatal to the cell for example, nucleases. However, synthesis
of proteins takes place on ribosomes in the cytoplasm, which presents two problems: in
order for protein synthesis to occur at all, m!"s must leave the nucleus where they are
transcribed, and also mature nuclear proteins must be transported into the nucleus. #he
processes of import and export are controlled to ensure the correct molecules are in the
correct cellular compartment.
Nuclear pore complex
#he nuclear envelope is studded with trans$membrane nuclear pore complexes, which
consist of % nucleoporin subunits surrounding a central channel through which proteins and
!"s can pass. &ibrils pro'ect from both surfaces of the nuclear pore complexes; those
inside the nucleus are organised as baskets.
(t has been shown by cryoelectron tomography that the central gate is occupied by
nucleoporin proteins rich in regular repeats of two hydrophobic amino acids, phenylalanine
and glycine, which interact with eachother and form a dynamic, saturated hydrogel. #his
hydrogel places a )nm limit on the effective diameter of the nuclear pore, which has an
actual diameter of *+nm. #he hydrogel restriction makes the pore too small for proteins
larger than ,+$*+k-a to passively diffuse through. .maller molecules however can diffuse
through the pore channel.
Import of nuclear proteins
!uclear proteins can accumulate in the nucleus in concentrations /++ times greater than in
the cytoplasm. #hrough experiments in which nuclear proteins were micro$in'ected into the
cytoplasm, it was proven that nuclear proteins re$accumulate in the nucleus, by selective
entry through the nuclear pore complexes. #he underlying mechanism was elucidated by
studying the import of the nuclear protein, nucleoplasmin, which accumulates in the nucleus.
!ucleoplasmin consists of two domains, a tail and a head. (n one experiment, whole, radio$
labelled nucleoplasmin molecules were microin'ected into the cytoplasm of a 0enopus
oocyte, and were shown to accumulate in the nucleus. (n two other experiments, first the
nucleoplasmin was sub'ected to limited proteolysis. #hen, either isolated nucleoplasmin
heads or tails, both radiolabelled, were microin'ected into the 0enopus oocyte cytoplasm.
#he tail domains were shown to be taken up into the nucleus, whereas the head domains
alone were not. #his suggested that the tail domain is necessary and sufficient for import into
the nucleus; a final experiment in which colloidal gold particles were coated with tail domains
and then microin'ected showed accumulation of gold into the nucleus via the nuclear pores,
detected by electron microscopy.
1roteins that localise to the nucleus, for example .2,+ large # antigen, contain small
peptide se3uences that are sufficient for import. 4hen fused to non$nuclear proteins, they
cause accumulation of the fusion protein in the nucleus. 5utations of certain lysine residues
of the .2,+ # antigen to threonine or asparagine abolish transport; certain amino acids of
this nuclear localisation signal are essential. (t should be noted that the nuclear localisation
signal is not like a signal peptide it is not cleaved, it is a part of the mature protein. #his is
important because the nucleus completely degrades in mitosis, and proteins must be re$
targeted once per cell cycle.
#ransport takes place in two steps: binding of the cargo to the cytoplasmic side of the
nuclear pore complex, followed by a slower, energy$dependent translocation through the
core. #wo proteins are essential for these steps: importin and ran. #he state of ran is
controlled by ran 6#1ase activating protein, which is present in the cytosol and causes ran
to convert from ran$6#1 to ran$6-1.
#he alpha subunit of importin binds to the nuclear localisation se3uence of the cargo. an$
6-1 is re3uired for cargo binding. #he beta subunit of importin docks at the nuclear pore
complex, associating with the hydrophobic repeats. "fter the alpha subunit importin has
been transported with the cargo through the nuclear pore, nuclear ran$6#1 causes the
cargo and the importin alpha subunit to dissociate. (mportin alpha and beta are exported
back into the cytoplasm separately, in a ran$6#1$dependent manner.
Export of proteins and RNA
!"s are exported bound to proteins. !" export is signal$dependent and carrier$mediated,
taking place through the nuclear pore complex. 7efore m!"s are exported, they must first
be correctly processed, including splicing and poly " tail addition.
!uclear export signals have been identified in several exported proteins, including those that
bind !"s. Export signals are recognised by exportin proteins, which are related to the beta
subunit of importin. .imilarly to import, regulation of export depends on the ran$6#18ran$
6-1 gradient across the nuclear envelope. an$6#1 binds to exportin in the nucleus,
causing a conformational change increasing its affinity for the cargo. 9nce bound, the
exportin$ran$cargo complex leaves the nucleus. :ytosolic ran$6"1 causes conversion of
ran$6#1 to ran$6-1, leading to the dissociation of exportin, ran and cargo. Exportin and
ran$6-1 are recycled separately to the nucleus, and guanine exchange factor converts ran$
6-1 to ran$6#1 in the nucleus.
(n conclusion, various proteins are important or necessary for nuclear import and export
importins interact with cytosolic ran$6-1 to control import, and associate with nuclear import
signals in the amino acid se3uence of cargo proteins. .imilarly, nuclear export is controlled
by exportins that interact with nuclear ran$6#1, and also with nuclear export signals in
proteins that bind !"s. #ransport regulation can have further impacts than simply ensuring
that essential proteins are properly localised. &or example, some transcription factors can
only be freed to enter the nucleus when dissociated from cytoplasmic anchors, adding an
extra layer of gene expression regulation. 1hosphorylation of nuclear import or export
signals may be another way of regulating nuclear transport for example, cyclin 7/ shuttles
between nucleus and cytoplasm in the . and 6; phases of the cell cycle, but
phosphorylation of a nuclear export signal in early mitosis blocks export, and results in
accumulation in the nucleus.