Lecture 38: Protein Trafficking and Nuclear Transport (good but look at c summary) Flashcards
Monday 20th January 2025
All eukaryotic cells have a basic set of membrane-enclosed organelles – compartmentation!
All eukaryotic cells have a basic set of membrane-enclosed organelles – compartmentation!
Nucleus…
site of DNA and RNA synthesis
Cytoplasm…
cytosol + organelles
Cytosol…
50% cell volume, site of protein synthesis and many metabolic pathways
Endoplasmic reticulum…
50-60% cell membrane, start point of secretory pathway
Golgi apparatus…
10% cell membrane, important for sorting and modifying proteins and lipids passing through it.
Lysosomes…
1% cell volume, multiple “suicide” bags for digestion of materials
Mitochondria/Chloroplasts (plants)…
25% cell volume, generate ATP
Peroxisomes…
1% cell volume, multiple sites for oxidative reactions
Plant vacuoles…
90% cell volume, for turgor or protein storage/ degradation
In 1999, what did Günter Blobel win a Nobel prize for?
He won the Nobel prize for the discovery that proteins have intrinsic signals that govern their transport and localisation in the cell.
In 2013, what did Jim Rothman, Randy Schekman, and Thomas Südhof win a Nobel prize for?
They won a Nobel prize for their discoveries of machinery regulating vesicle traffic, a major transport system in our cells.
What are the different modes of protein transport?
Gated transport
Protein translocation
Vesicular transport
What is gated transport?
import into and export out of the nucleus.
What is protein translocation?
protein import into ER
What is Vesicular transport?
secretion along the organelles of the secretory pathway
what so proteins that don’t reside in the cytosol need in order for them to reach their location?
They need sorting signals.
Are sorting signals part of a protein’s sequence?
Yes
What can sorting signals be?
- Short peptides at the N- or C-termini, which can be removed after use or kept on if needed again (e.g. for nuclear transport).
- 3-dimensional domains (secondary/tertiary structure) e.g. for transport to lysosomes
- Other molecules attached to the protein (post-translational modifications) such as sugars and lipids.
What happens to sorting signals?
- They’re recognised by specific receptors within the cell
- This in turn triggers the transfer of the protein to the correct destination
- Every organelle uses different receptors and different sorting processes
- If any of this processes goes wrong, the cell is in big trouble (there’s several diseases associated with the mis-sorting of proteins).
- Can be removed after use or kept on if needed again
How do proteins and other macromolecules (e.g. ribosomes) move between the cytoplasm and nucleus (in and out)?
Via large aqueous nuclear pore complexes (NPC).
How many nuclear pore complexes does a mammalian nuclear pore complex (NPC) contain?
A mammalian nuclear envelope contains 3000-4000 nuclear pore complexes (NPCs)
Describe the structure of a nuclear pore complex
- Eightfold radial symmetry, embedded in the nuclear envelope.
- Composed of ~30 nucleoporins (Nups) forming distinct regions:
- Cytoplasmic & Nuclear Rings – Anchor NPC; cytoplasmic filaments aid cargo recognition.
- Central Transport Channel – FG-nucleoporins create a selective sieve.
- Nuclear Basket – Extends into nucleoplasm, regulates cargo.
- Membrane-Embedded Scaffold – Anchors NPC to the nuclear envelope.
How large are ribosomes?
3,300kDa
How large are nucleoporins?
Each NPC is 125,000 kDa (30 times bigger than a ribosome) and is made up of many copies (16 or multiples of 16) of ~30 different nucleoporins
‘Small molecules diffuse extremely fast (almost free flow) through Nuclear Pore Complexes’. Is this true?
Yes
What would a small molecule be classed as?
5 kDa or less)
How quickly do proteins of 20-40 kDa diffuse?
They diffuse a lot more slowly
Can proteins larger than 40kDa diffuse freely through a nuclear pore complex?
No, and instead, they move through active transport.
What do proteins that are larger than 40kDa carry?
They carry nuclear localisation or export signals.
What is the diffusion barrier caused by?
The diffusion barrier is caused by unstructured (disordered) regions of channel nucleoporins rich phenylalanine-glycine (FG) repeats forming a tangled network (mesh, or matrix), blocking the passive diffusion of large molecules (e.g. ribosomes).
Describe nuclear localisation signals (NLS)
- NLS are rich in lysine and Arginine (+ charged), and can be in any position of the passenger (= cargo) protein, so long as they are exposed to the surface of the protein
- So long as they are exposed to the surface of the protein
- Nuclear Localisation Signals are recognised by a family of cytosolic nuclear import receptors (importins or karyopherins) – each member being responsible for a set of cargo molecules (or sometimes a cargo adaptor
How do FG repeats facilitate active transport through the Nuclear Pore Complex (NPC) ?
- Nucleoporins with FG repeats form the mesh
- Import receptors have low affinity binding sites for the FG repeats
- FG repeats in cytosolic fibrils help recruit the receptors and bind the cargo
- This complex then travels through the pore mesh through transient interactions
- The receptor disengages from the cargo protein on the other side
Gated transport – ACTIVE nuclear import
ACTIVE how?
- Moving certain proteins inside the nucleus creates different protein pools in the nucleoplasm compared to the cytoplasm
- This type of nuclear transport creates order in the cell
- ORDER = energy required to maintain it
- The energy for this type of transport comes from GTP via the GTPase-Ran
What does the energy for the active transport come from?
The energy for this type of transport comes from GTP via the GTPase-Ran
The GTPase Ran..
- GTPase ‘switch’:
- Hydrolyses GTP to GDP (binds both)
- Conformation is different depending if bound to GTP or GDP
- different conformation = different activity
In the nucleus: RanGTP binds importins, causing them to release cargo.
In the nucleus: RanGTP binds importins, causing them to release cargo.
‘Ran-GTPase provides both the free energy and the directionality for nuclear transport’. Is this true?
Yes
What is the net import of cargo driven by?
Net import of cargo is driven by two conformational states of the GTPase Ran, that depend on whether Ran binds GDP or GTP
Ran-GDP…
Ran-GDP has conformation (A) and is in this state only in the cytosol
Ran-GTP…
Ran-GTP has conformation (B) and is in this state only in the nucleus
What is Ran-GTPase regulated by?
Ran-GTPase is regulated by GAP and GEF
What is GAP?
GTPase activating protein (promotes hydrolysis of GTP to GDP)
What is GEF?
guanine nucleotide exchange factor (exchanges GDP with GTP)
Nuclear import (ACTIVE TRANSPORT): summary
- GTPase activating protein
GAP ensures that Ran-GDP is generated, and in this state, Ran falls off the importin - Facilitated transport via low affinity binding or IMPORTIN to FG repeats
- Guanine Nucleotide Exchange factor
GEF ensures that Ran-GTP is generated, and this prefers to bind to importin, displacing the imported protein
Unlike other signal peptides for protein sorting, the NLS is not cleaved after import because:
- it is often needed again. This is because many nuclear proteins constantly cycle to the cytosol, so they must be repeatedly imported-
- re-import must also happen after every mitosis when the nucleus reforms
- the NLS may be internal and part of a functional domain
-
Is it true that nuclear export is like nuclear import, but in reverse?
Yes
What are Nuclear export signals (NES) ‘seen’ by?
Nuclear export signals (NES) are “seen” by soluble nuclear export receptors - exportins.
Exportins…
- Exportins (related to importins) bind the NES and the FG-nucleoporins in the channel and filaments, to move the cargo to the cytosol.
- and use Ran in reverse (RanGDP releases cargo in the cytosol)
How can we visualise subcellular organelles in vivo?
We can visualise subcellular organelles in vivo using dyes or Fluorescent Proteins such as GFP.
Is nucleo-cytoplasmic trafficking reversible?
Yes
What are other roles of NPCs?
- NPCs also control genome regulation and ageing:
- Nup98 activates genes in the context of acute myeloid leukemia
- Some Nups have a very long half-life (several years!), so they deteriorate with age and affect cellular health
SUMMARY
- Proteins need signals to travel to their correct intracellular destination
- Sorting signals are found within the protein and need to be exposed e.g. nuclear proteins require NLS
- Cytoplasm – nucleoplasm traffic:
- Occurs through the nuclear pore complexes (NPCs)
- Uses receptors called importins (into the nucleus) or exportins (out of the nucleus)
- Directionality of movement is provided by the Ran GTPase switch
- Asymmetric distribution of Ran GTP (nucleus) and Ran GDP (cytosol) depends on the Ran GAP (cytosol) and Ran GEF (nucleus)
- Asymmetric distribution of Ran GAP and Ran GEF depends on their preferential association with the cytosolic cytoskeleton and nuclear chromatin, respectively
