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Fig. 12-17
Deamination
Fig. 2-15
Phosphorylated CTD
U1snRNP
Cross-exon recognition
complex
U2snRNP
Pol II
nascent pre-mRNA
hnRNP
proteins
mRNP
CBP
hnRNP proteins
Splicing
CBP
Cleavage/
polyadenylation
AAAAAAAAAAA
PABPII
Fig. 5-19
Fig. 5-22
Nuclear pore complexes are made of multiple copies of
~100 different proteins.
The general term for one of the proteins that make up the
nuclear pore complex is “nucleoporin.”
The specific name for a nucleoporin is generally based on
its molecular weight, such as “Nup 150.”
NPCs are roughly octagonal, membrane-embedded
structures from which eight ~100 nm fibers made of
specific Nups extend into the cytoplasm. Similarly, eight
~100 nm fibers extend into the nucleoplasm where there
ends are connected, forming a “nuclear basket.”
Fig. 12-18
Fig. 12-18
The nucleoplasmic side of an NPC is attached to an
orthagonal network of lamin intermediate filaments called
the “nuclear lamina.”
This nuclear lamina supports the inner nuclear membrane,
giving strength and shape to the nuclear envelope.
Fig. 21-16
Ions, small metabolites, and small proteins (< ~60 kDa)
can diffuse through a water-filled channel in the NPC.
However, large proteins and mRNPs cannot diffuse
freely in and out of the nucleus. They must be
selectively transported out of or into the nucleus with
the aid of soluble transporter proteins called “importins”
or “exportins” or “karyopharins.” These karyopharins
bind to short peptide sequences in the proteins to be
transported and to nucleoporins to transport their
“cargo” proteins through the central transporter of the
NPC.
A class of nucleoporins called FG-nucleoporins line the
channel in the NPC central transporter and are also found
in the cytoplasmic and nucleoplasmic filaments.
FG-nucleoporins contain domains with repeats of multiple
phenylalanine (F) and glycine (G) amino acid residues
separated by stretches of hydrophilic amino acids.
The domains of FG nucleoporins containing the FG
repeats are postulated to form extended polypeptide
strands with hydrophobic repeats punctuating the extended
strands.
Hydrophilic
region
FG-repeat
These domains are postulated to extend into the channel
of the central transporter where the hydrophobic FG
repeats from separate FG-nucleoporins interact forming a
molecular meshwork.
K. Ribbeck and
D. Gorlich (2001)
EMBO J. 20:1320
This molecular meshwork in the
central transporter of the NPC is
postulated to allow small proteins
to diffuse through the meshwork,
but to prevent large proteins and
mRNPs from diffusing through.
SV40 T-antigen nuclear localization signal -- NLS: PKKKRKV
Pyruvate kinase
Fig. 12-19
Pyruvate kinase-PKKKRKV
All large nuclear proteins studied contain a
peptides sequence of < 50 aa that cause most
soluble cytoplasmic proteins to be transported
into the nucleus when fused to that protein.
Many of these NLSs are rich in lysine (K) like the
SV40 NLS.
But other NLSs have a variety of sequences that
are not K-rich.
A digitonin-permeabilized cell system provided an assay
for cytoplasmic proteins required for the transport into
nuclei of proteins with an NLS. This non-ionic detergent
makes wholes in the plasmamembrane that allows
cytoplasmic proteins to leak out of the cell without
disrupting the nuclear membrane.
Proteins added to these digitonin-treated cells can come
in contact with the intact nuclear envelope.
If a fluorescently labeled protein with an NLS was added
to these digitonin treated cells, it did not accumulate in the
nucleus.
However, if a cytoplasmic protein extract was added to the
cells along with the fluorescently labeled protein, it did.
Fig. 12-20
Using the transport of a fluorescently labeled protein
with an NLS into digitonin permeabilized cells as an
assay for the soluble cytoplasmic proteins required
for nuclear transport, only four proteins were found
to be required:
Ran a small G protein similar to Ras.
Importin a, Importin b
Nuclear transport factor 2 -- NTF2
These domains are postulated to extend into the channel
of the central transporter where the hydrophobic FG
repeats from separate FG-nucleoporins interact forming a
molecular meshwork.
K. Ribbeck and
D. Gorlich (2001)
EMBO J. 20:1320
This molecular meshwork in the
central transporter of the NPC is
postulated to allow small proteins
to diffuse through the meshwork,
but to prevent large proteins and
mRNPs from diffusing through.
SV40 T-antigen nuclear localization signal -- NLS: PKKKRKV
Pyruvate kinase
Fig. 12-19
Pyruvate kinase-PKKKRKV
All large nuclear proteins studied contain a
peptides sequence of < 50 aa that cause most
soluble cytoplasmic proteins to be transported
into the nucleus when fused to that protein.
Many of these NLSs are rich in lysine (K) like the
SV40 NLS.
But other NLSs have a variety of sequences that
are not K-rich.
A digitonin-permeabilized cell system provided an assay
for cytoplasmic proteins required for the transport into
nuclei of proteins with an NLS. This non-ionic detergent
makes wholes in the plasmamembrane that allows
cytoplasmic proteins to leak out of the cell without
disrupting the nuclear membrane.
Proteins added to these digitonin-treated cells can come
in contact with the intact nuclear envelope.
If a fluorescently labeled protein with an NLS was added
to these digitonin treated cells, it did not accumulate in the
nucleus.
However, if a cytoplasmic protein extract was added to the
cells along with the fluorescently labeled protein, it did.
Fig. 12-20
Using the transport of a fluorescently labeled protein
with an NLS into digitonin permeabilized cells as an
assay for the soluble cytoplasmic proteins required
for nuclear transport, only four proteins were found
to be required:
Ran a small G protein similar to Ras.
Importin a, Importin b
Nuclear transport factor 2 -- NTF2
K. Ribbeck and
D. Gorlich (2001)
EMBO J. 20:1320
= FG repeat
Importin
NTF2 binds Ran•GDP and interacts with FG-repeats.
NTF2 does not bind Ran•GTP.
+
NTF2
+
NTF2
Fig. 12-21
The direction of transport is dependent on the localization
of Ran-GAP in the cytoplasm and Ran-GEF in the nucleus.
This maintains Ran in the GTP form in the nucleus and in
the GDP form in the cytoplasm.
Ran-GAP is bound to the cytoplasmic filaments of the NPC.
Ran-GEF is bound to chromosomes.
Fig. 12-22
Nuclear
mRNP
PABPII
Nucleus
Nucleus restricted
hnRNPs
Nuclear
pores
Cytoplasm
hnRNP
Protein
shuttle
Exchange of nuclear mRNPs proteins
for cytoplasmic mRNP proteins
Cytoplasmic
mRNP
PABPI
Cytoplasmic mRNP proteins
Fig. 12-23