Nucleus and nuclear import/export Flashcards
Nuclear pore complex 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
- core scaffold provides stability and set the size of NPC
- sub complexes show 8 fold symmetry
- Nups in core scaffold are very long lived and diminish in aging cells
What anchors the nuclear pore complex (NPC) in the nuclear envelope?
Luminal ring
- Nups in luminal ring are transmembrane proteins (not well conserved across species)
What provides a selective permeability barrier in the nuclear pore complex?
the central pore
- Nups in the central pore and highly dynamic and contain FG repeats ( small, hydrophobic segments)
What are the principles for fast selective transport for nuclear pore complexes?
- diffusion-mediated transport of small molecules
- selective transport of larger cargo
- weak and flexible barrier for efficiency and to prevent clogging
What are FG repeats?
hydrophobic phenylalanine-glycine repeats
How do the centra pore Nups form a selective permeability barrier?
- FG repeats that are intrinsically disordered and highly dynamic
- FG repeats are hydrophobic but connected by flexible hydrophilic linkers
- creates a flexible, hydrophobic environment inside the pore
How does size-filtering diffusion work for nuclear pore complexes?
- driven by diffusion of hydrophilic molecules between the FG repeats inside the pore
- size less than or equal to 40 kDa (water, ions, smaller molecules)
How does spontaneous migration work for nuclear pore complexes?
- amphiphilic proteins (both hydrophobic and hydrophilic) that adjust to show more hydrophobic surfaces, allowing them to interact with FG Nups and pass through
- largest example is Dystrophin (427 kDa)
How does facilitated transport work for nuclear pore complexes?
- large, hydrophilic cargoes selectively bind to hydrophobic transport receptors, which facilitate interactions with FGs
- 230 kDa or more (most RNAs and proteins)
Principles of facilitated transport in nuclear pore complexes
- selectivity - cargoes selectively bind to amphiphilic transport receptors
- cargoes do not unfold - they remain hydrophilic but transport receptors unfold to facilitate migration
- directionality - cargoes are efficiently targeted for nuclear import or export
- transport does not require energy (energy is stabilizing or destabilizing the cargo and transport receptor)
What are nuclear pore complexes?
- transverse the double membrane of the nuclear envelope creating aqueous channels that are freely permeable to small molecules and ions but restrict passage of larger macromolecules
- stabilize sites of membrane fusion and promote fast and selective transport
Describe how transport receptors work with nuclear pore complexes
- cargo proteins contain specific amino acid motifs that are recognized by transport receptors
- transport receptors are necessary for transport and sufficient to cause transport
- 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 can be re-used
What are karyopherins?
- cargo receptors for nuclear import/export
- 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
What is the structure of the nuclear envelope?
- double bilayer, 30-50 nm between inner and outer membranes
- Outer Nuclear Membrane (ONM) is continuous with rough ER
- inner and outer membranes fuse at circular pores
What protein coordinates between the inner and outer nuclear membrane?
KASH (on outer nuclear membrane) connected to SUN (on inner nuclear membrane)
- stabilizes the nuclease
What do proteins on the Inner Nuclear Membrane do?
- attach to nuclear lamins (cytoskeletal filaments important for shape and structure)
- attachments for chromatin (organize)
- e.g. SUN
What does Ran-GTPase do?
- regulates the directionality of transport
- GTP form either promote dissociation (import) or stabilize complex formation (export)
Describe the nuclear import cycle.
- import karyopherin (importins only bound to cargo or RanGTP) binds to NLS cargo
- GEF dissociates the cargo and RanGTP promotes dissociation by binding to the karyopherin
- the RanGTP-karyopherin goes into the cytoplasm
- GAP hydrolyzes to RanGDP and it dissociates from karyopherin
Describe the nuclear export cycle.
- RanGTP promotes association of the cargo with the karyopherin (exporting - bound to both cargo and RanGTP)
- exported into the cytoplasm
- GAP breaks complex and RanGTP is hydrolyzed to RanGDP
- the karyopherin passes back in to the nucleus alone
Is mRNA nuclear transport the same as for proteins?
No, it uses ATP
Describe disruption of BRCA2-RAD51 nuclear transport.
BRCA2 is a common breast cancer mutation that unmasks its NES, causing it to be exported from the nucleus and impairs DNA damage response
What is GEF doing in the nucleus?
making RanGTP that binds to importins (release cargo) or exportins (bind to cargo)
What does GAP do in the cytoplasm?
activates GTPase activity (GTP -> GDP “off”)
