Nucleus & Nuclear Import & Export Flashcards
- Describe the structure of the nuclear pore complex.
Nuclear Pore Complex (NPC):
-stabilizes sites of membrane fusion and promote fast and selective transport
-multi-protein assemblies that form gated channels
across the nuclear envelope.
•Like little holes occupied by this NPC
•Think of it as
1) stabalzing fusion sites of inner and outer membranes;
2) PROVIDE a highly regulated gating system
- Density: HeLa cells = 11 NPCs/mm2 (~10% of NE surface); Xenopus oocyte = 50 NPCs/mm2 (~50% of NE surface); in short diff cells will have diff distribution
- Distribution along nuclear envelope is not random. Thought to be affected by nuclear lamina and chromatin
- Density is greater (~3X in some cells) than minimum requirement for nuclear transport, and decreases in aging cells. –> in yeast; over abundance decreases with age; NPC proteins live very long
- Describe the basic mechanisms of nuclear import and export of proteins and RNA/protein complexes, and how they can be regulated.
Transport Mechanisms:
1)Size-filtering diffusion:
•Examples: Water, ions, smaller molecules
•Size of Cargo: ≤ 40kDa ; that is the cut off
•Mechanism: Driven by diffusion of hydrophilic molecules between the FG repeats inside the pore.
2)Spontaneous Migration:
•Examples: β-catenin, Spectrin repeat proteins, karyopherins
•Size of Cargo: ??? Largest example is Dystrophin (427kDa)
•Mechanism: Amphiphilic cargoes undergo conformational changes to expose hydrophobic surfaces, allowing them to interact with FG Nups and pass through. Have amphipathic nature
3)Facilitated Transport:
•Examples: Most RNAs and proteins
•Size of Cargo: 230kDa or more
•Mechanism: Large, hydrophilic cargoes selectively bind to hydrophobic transport receptors, which fa
•Cargo maintain structure as they pass through, no conformational change
- Selectivity. Cargoes selectively bind to amphiphilic transport receptors.
- Cargoes do not unfold. Cargoes remain hydrophilic, but transport receptors unfold to facilitate migration.
- Directionality. Cargoes are efficiently targeted for nuclear import or export.
- Transport does not require energy
Nuclear export of mRNAs:
mRNA transport differs from protein
•Uses non-karyopherin transport receptors; Nxf1 and Nxt1.
•Does not use Ran.
•Directionality is conferred by RNA helicase (Dbp5) that remodels mRNAs as they exit the NPC.
Nuclear export of ribosomes:
•Most abundant and among the largest nuclear transport cargoes.
Similar themes, but requires multiple transport factors.
• For 60S ribosomal subunit, NES is provided by export adapter protein Nmd3, which binds to the assembling 60S subunit in the nucleus and is removed by a GTPase upon entering the cytoplasm.
• Remains poorly understood…
- Explain how changes that impact nuclear transport and/or NPC components can contribute to disease.
Transport defects in disease:
•Viral infection. Many viruses target Nups and alter NPC structure to gain access to the nucleus.
•Cell Proliferation and Tumorigenesis. Many oncogenes and tumor suppressors are regulated by nucleocytoplasmic shuttling.
Example): Disruption of BRCA2-RAD51:
BRCA2D2723H is a common breast cancer mutation that unmasks its NES, causing it to be exported from the nucleus. This impairs DNA damage response. BRACA is out of nucleus cant do repair needed for cancer
Nup gene fusions involved in cancer:
•TPR – fuses to and hyperactivates kinases MET (gastric cancer), NTRK1 (thyroid carcinoma), FGRF1 (myeloproliferative syndromes), ALK (lung adenocarcinoma).
- RANBP2 – fuses to ALK kinase and mis-localizes it to NPC core; found in myofribroblastic tumors, myeloid malignancies, and large B-cell lymphoma.
- NUP214 – fuses to oncogenes SET and DEK and sequesters them to accumulations in the nucleus in AML and T-cell acute leukemias.
- NUP98 - at least 32 different fusions identified in hematological malignancies. Very well known
- Describe the structure of the nuclear pore complex and roles of:
- nucleoporins (Nups)
Nucleoporins (Nups):
-protein subunits of the nuclear pore complex
-Although NPCs are one of the largest proteinaceous structures in the cell (with a typical mass of ~125 MDa) they are composed of only ~30 distinct proteins (collectively called
nucleoporins, or Nups).
NPC Major Structural Features:
•500-1000 total protein subunits
•Made from ~30 distinct proteins known as Nucleoporins (Nups)
•Form stacked ring structure with a 40nm central channel
•Images change from top to bottom; diff layout depending at what level your at: z palne view
1)Core Scaffold:
(scaffolding) backbone
Core scaffold provide stability and set the size of the NPC.
• Subcomplexes show 8-fold symmetry. Has an outer and inner ring
• Nups in core scaffold are very long lived (months to years), and diminish in aging cells.
2)Lumenal Ring: (membrane anchored)
-embeds it to nuclear membrane;
oLuminal ring anchors the NPC to the nuclear envelope. Allows for bending of membrane locally; really links core scaffold to the membrane
oNups in luminal ring are transmembrane proteins.
oThese Nups are not well-conserved across species
3)Central Pore: (barrier nups)
The barrier Nups contain regions that are enriched in phenylalanine/glycine (FG) repeats.
These are intrinsically
disordered domains that together create an entropic barrier to the passage of
hydrophilic macromolecules.
oCentral pore forms a selective permeability barrier
oNups in the central pore are highly dynamic and contain FG repeats
oFG repeats are AA residues, Nups that line central pore have many FG repeats.
oNup 116 have FG repeats that are separated by hydrophobic areas; think of it like disordered tentacles FG repeats = phenylalanine-glycine repeats • Many repeats in each protein, total of ~3500/NPC. • Intrinsically disordered and highly dynamic. • FG repeats are hydrophobic, but connected by flexible hydrophilic linkers. • Creates a flexible, hydrophobic environment inside the central pore. –>form selective permeability repeats
oControversy in the NPC field: Do the FG Nups form a highly crosslinked ‘hydrogel’? -OR Are the FG Nups structured into layers that promote active transport?
- Describe the structure of the nuclear pore complex and roles of:
- karyopherins
Karyopherins:
Are cargo receptors for nuclear import/export; they are amphiphilic.
•Karyopherins are amphiphilic proteins that bind to NLS or NES.
•Binding causes conformational change that exposes hydrophobic surfaces.
•Importins transport into the nucleus.
- Exportins transport out of the nucleus.
- Cells express different importins and exportins with different cargo selectivity
Wait wtf are NLS & NES? Good question!:
Selectivity is mediated by cargo transport signals: NLS = in (import), & NES = out (export)
•Cargo proteins contain specific amino acid motifs that are recognized by transport receptors. These are necessary and sufficient.
•Nuclear Localization Sequences (NLS) bind to receptors that facilitate transport into the nucleus.
•Nuclear Export Sequences (NES) bind to receptors that facilitate transport out of the nucleus.
•Signal motifs are not cleaved, and can be re-used made up of basic residues like lysine and arginines; NES is rich in hydrophobic leucines
(from handout)
-proteins that facilitate transport through the nuclear pore complex by
selectively binding to cargoes and interacting with nucleoporins inside the central channel of the nuclear pore complex.
- Describe the structure of the nuclear pore complex and roles of:
- Ran in transport.
Ran:
Regulates the directionality of transport.
•Ran stabilizes cargo-karyopherin complex.
•Ran is GTPase enzyme (similar to Ras) that acts as a molecular switch; interactions with karyopherins depends on its nucleotide status.
•Nucleotide status is regulated by GAP (cytoplasm) and GEF (nucleus) proteins; this creates a gradient of Ran activity across the NPC.
- In nucleus, Ran tends to be bound to GTP, which stabalzies cargo interactions with karopherins that drive export from the nucleus
- In cytoplasm, Ran encounters a regulator (GAP) that stimulates its hydrolysis activity–> this drives it into the GDP state; decreases its affinity for the cargo-karopherin complex and breaks it apart and thats the end transport; thats how you maintain the directionality
(from handout)
-small GTPase enzyme that regulates transport through nuclear pore complexes
by altering interactions between cargoes and karyopherins.
and while your at it tell me what the nuclear envelope is
why is this shit sooo boring
Nuclear Envelope (NE):
- membrane barrier separating the nucleus from the cytoplasm
- separating the genome from the cytoplasm
- composed of two essentially concentric lipid bilayers.
- ONM is continuous with rough ER
- ONM proteins are involved in positioning the nucleus and creating attachments to the inner membrane; KASH on slides
- The outer and inner membranes fuse at circular pores
- INM proteins create attachments to proteins on the outer membrane (SUN mother fucker) as well as attach to nuclear lamins and chromatin
- so essentially SUN and KASH bind to each other those tethering the outer membrane to the inner
- Also of importance is the relationship of the Inne INM proteins to other structures within the nucleoplasm:
- Emerin: loss of function mutations cause X-linked Emery-Driefruss Muscular Dystrophy. Mutatnt cells show aberrant nuclear shape and increased apoptosis.
- LMNA (Lamina A): splice mutants (nonsense mutation, truncated protein) cause Hutchinson-Gilford progeria in which mutatnt cells show defective nucelar shape, chromatin organization, genome stability and other phenotypes.
