import and export of nucleus Flashcards
The structure of the nuclear pore complex (NPC) consists primarily of a spoke complex:
It has an ______
octagonally symmetric cylindrical structure about 50nm tall around the axis of transport and a planar pseudo-symmetry through the nuclear envelope
Attached to the central framework are
8 cytoplasmic filaments and a nuclear basket formed from 8 filaments that join into a distal ring.
The linear dimensions of the NPC vary between species, but the ______ is conserved.
3D architecture
NPC External diameter is about
125nm
The ______ are a family of proteins which are the constituent building blocks of the NPC.
nucleoporins
_______ are repetitively arranged in distinct subcomplexes.
About 30 distinct nucleoporins (Nups)
There are a total of _____ per NPC.
500-1000 nucleoporins
A typical nucleoporin has a domain structure with an N terminus containing _______ and a C terminus rich in ______
multiple copies of the motif XFXFG (FG repeats)
heptad repeats characteristic for coiled-coil conformation.
a typical nucleoporin has two domains
N and C terminus
There are several models for the NPC permeability barrier to cargo, all of which involve _______:
(4)
FG Nups
- Selective Phase/Hydrogel Model
- Virtual Gate/Polymer Brush Model
- Forest Model
- Reduction of Dimensionality Model
Selective Phase/Hydrogel Model
Barrier is a 3-D sieve formed from inter- and intra-molecular interactions of FG-Nups
Virtual Gate/Polymer Brush Model
FG-Nups move vigorously back and forth to create a repulsive barrier with few stable
cohesive contacts
Forest Model
- FG-Nups adopt a mixture of extended (“tree”) and collapsed (“shrub”) conformations.
- The ends of several “trees” can form cohesive contacts to create a central tunnel
(zone 1) that acts as a hydrogel, with a second route closer to the walls
(zone 2) acting as a virtual gate
Reduction of Dimensionality Model
- FG-Nups coat the inner surface of the pore to form a hydrophobic surface
- Hydrophilic unfolded chains extend into the central pore to create a selectivity filter
Recent work has shown that nucleoporins pull double duty as.
transcription factors regulating the activity of genes active during early development
Nucleoporins don’t regulate all genes, but are required for a subset of genes, including
developmentally regulated genes, which are turned on and off in a controlled manner during cell differentiation and tissue development.
NPC components:
The gene doesn’t go to the pore; the pore protein goes to the gene.
_______ may also be a potential marker for cancer.
Nucleoporins
Nucleoporins ______are highly overexpressed in some cancers, including _______.
Nup214 and Nup88
colon cancer and very aggressive forms of lung cancer
Karyopherins are the proteins that
the proteins that actually facilitate nuclear transport through the NPC. The act as mediators between the nuclear pore complex and the cargo being transported.
Karyopherins may act as
importins or exportins, and are part of the Importin-β super-family which all share a similar three-dimensional structure.
Karyopherins are ______ molecules composed ______
heteromeric
two major types of components, alpha karyopherins and beta karyopherins, that function together to transport molecules through the NPC.
Beta-karyopheryins
receptor family of karyopherins
directly interact with FG Nups
Some also interact directly with cargo
Alpha karyopheryins
adaptor family of karyopherins
facilitate cargo selective binding
Several other proteins such as _____ and _____ bind to karyopherins and participate in the transport process.
Ran GTP binding protein and cellular apoptosis susceptibility protein
Ran is a:
(RAs-related Nuclear protein)
small 25Kda protein that is involved in transport into and out of the cell nucleus.
Ran is a member of the ____ family
Ras superfamily.
Ran is a ______ protein that is essential for the ______
GTP binding
translocation of RNA and proteins through the NPC.
Ran exists in the cell in two nucleotide-bound forms:
GDP-bound and GTP-bound.
RanGDP is converted into RanGTP through the action of
regulator of chromosome condensation 1
(RCC1), the nucleotide exchange factor for Ran.
Ran’s intrinsic GTPase-activity is activated through interaction with ______, facilitated by complex formation with _____
Ran GTPase activating protein (RanGAP),
Ran-binding protein (RanBP).
GTPase-activation leads to the conversion of _____, thus closing the Ran cycle.
RanGTP to RanGDP
Ran can diffuse freely within the cell, but because RCC1 and RanGAP are located in different places in the cell, the concentration of
RanGTP and RanGDP differs locally as well, creating concentration gradients that act as signals for other cellular processes.
RCC1 is located ______
inside the nucleus
RanGAP is bound to _____
the nuclear envelope I
This difference in location of the accessory proteins in the Ran cycle leads to
a high RanGTP to
RanGDP ratio inside the nucleus and an inversely low RanGTP to RanGDP ratio outside the nucleus
RanGTP =
nuclear export
In addition to a gradient of the nucleotide bound state of Ran, there is a gradient of the protein
itself, with a higher concentration of_____
Ran in the nucleus than in the cytoplasm
Cytoplasmic RanGDP is imported into the nucleus by _______
the small protein NTF2 (Nuclear Transport Factor 2),
in the nucleus, RCC1 can then catalyze exchange of______
GTP for GDP on Ran
Ran is involved in the transport of
proteins across the nuclear envelope by interacting with karyopherins and changing their ability to bind or release cargo molecules.
Cargo proteins containing a NLS are bound by
importins and transported into the nucleus o Inside the nucleus, RanGTP binds to importin and releases the import cargo
Cargo that needs to get out of the nucleus into the cytoplasm binds to
exportin in a
ternary complex with RanGTP
Upon hydrolysis of RanGTP to RanGDP outside the nucleus, the complex
dissociates and export cargo is released
Nuclear localization signal and human disease:
- Male to female sex reversal
2. Cowden Syndrome
Male to female sex reversal –
- XY female with external feminine genitalia but a lack of formation of both ovaries and testes
- Normally Importin Beta 1 binds to SRY to translocate to the nucleus, where it can
upregulate transcription of target genes - If SRY lacks an NLS, it will not be translocated to the nucleus and male sex determining
genes will not be transcribed, resulting in male to female sex reversal
Cowden Syndrome
- Autosomal dominant disease with high susceptibility to multiple hamartomas affecting
derivatives of all three germ layers and by an increased risk of breast, thyroid, and
endometrial neoplasia - Protein of interest is PTEN, which is normally translocated by Kapβ o If no NLS, will not enter nucleus
Nup Fusions in Cancer:
- NUP98/HOX fusions in acute myeloid leukemia (AML)
2. Aberrantly induces transcription of downstream HOX genes
Role of NPC in Aging:
- No new NPCs are produced in post-mitotic cells
- This makes them prone to oxidative damage
- The result is a leaky nuclear pore
- This is seen in aging
inhibiting nuclear translocation of calcineurin can prevent
myocardial hypertrophy.
Nucleoporin: Human Nup 155
Tissue specific disorder:
Af, which is a cardiac disorder characterized by clinical arrhythmia
Nucleoporin: Human Nup98
Tissue specific disorder:
Acute myeloid leukemia (and other hematological malignancies)
haematopoeitic stem cell proliferation
Nucleoporin: Human Nup62
Tissue specific disorder:
primary bilary cirrhosis (autoimmune liver degen) for autoimmune antigen mutation
OR
Aitsomal recessive infantile bilalteral striatal necrosis (degeneration of the basal ganglia) for the homozygous missense Q391P mutation
Nucleoporin: Human Nup358/RanBP2 (-)
Tissue specific disorder:
Familial cases of infection triggered acute necrotizing encephaly
Nucleoporin: Human Nup 155
Mutation:
homozygous missense R391H
Nucleoporin: Human Nup98
Mutation
multiple genomic translocations, which results in fusion of Nup98 fragment, including FG repeats to another gene
Nucleoporin: Human Nup 62
Mutation:
autoimmune antigen or homozygous missense Q391P mutation
Nucleoporin: Human Nup358/RanBP2 (-)
mutation:
heterozygous missense mutation T585M
three layers of the NPC
- membrane
- scaffold
- barrier
the layer that is functional s the
barrier layer. this acts as the selective gate
the scaffold layer provides the
curvature of the NPC
_____ in, ____ out
NLS
NES
Ran.GTP:Importin/Exportin binding is _____, and blocks ______;
_____ relieves this block allowing ______
very tight
Ran’s intrinsic GTPase activity
RanBP1
RanGAP1 to activate Ran’s intrinsic GTPase by up to 105 fold
_____ not cleaved during transport, so can be re-used
NLS/NES
The translocation channel is a proteinaceous pore lined by
natively unfolded proteins that use hydrophobic interactions to form a selective filter.
ImporVn α an _____
adapter protein:
> 7 family members in humans
ImporVn β the
cargo carrier: >20 family members involved in both import and export, many members do not require an adapter protein
Proteins (or protein complexes) may shuttle: contain both
NLS and NES
Ran accounts for ___% of total cell protein
0.4% total cell protein ≈107 copies/cell
The signals are typically exposed linear sequences of amino acids, often containing _____
multiple basic and/or hydrophobic residues.
They are not cleaved following transport.
cargo transporters are generally members of the ____ family.
karyopherin
Transport is regulated by the
- entropy barrier created by the Nups,
- the presence of the Ran.GDP/GTP gradient,
- the interaction of cargo transporters with nucleoporins, and
- cargo modifications/interactions that impact association with receptors.
Diseases occur when proteins are
mislocalized, either because the selectivity barrier is compromised, or mutations perturb key interactions.
