Lecture 38: Protein Trafficking and Nuclear Transport (good but look at c summary)

Question 1

All eukaryotic cells have a basic set of membrane-enclosed organelles – compartmentation!

Answer 1

All eukaryotic cells have a basic set of membrane-enclosed organelles – compartmentation!

Question 2

Nucleus…

Answer 2

site of DNA and RNA synthesis

Question 3

Cytoplasm…

Answer 3

cytosol + organelles

Question 4

Cytosol…

Answer 4

50% cell volume, site of protein synthesis and many metabolic pathways

Question 5

Endoplasmic reticulum…

Answer 5

50-60% cell membrane, start point of secretory pathway

Question 6

Golgi apparatus…

Answer 6

10% cell membrane, important for sorting and modifying proteins and lipids passing through it.

Question 7

Lysosomes…

Answer 7

1% cell volume, multiple “suicide” bags for digestion of materials

Question 8

Mitochondria/Chloroplasts (plants)…

Answer 8

25% cell volume, generate ATP

Question 9

Peroxisomes…

Answer 9

1% cell volume, multiple sites for oxidative reactions

Question 10

Plant vacuoles…

Answer 10

90% cell volume, for turgor or protein storage/ degradation

Question 11

In 1999, what did Günter Blobel win a Nobel prize for?

Answer 11

He won the Nobel prize for the discovery that proteins have intrinsic signals that govern their transport and localisation in the cell.

Question 12

In 2013, what did Jim Rothman, Randy Schekman, and Thomas Südhof win a Nobel prize for?

Answer 12

They won a Nobel prize for their discoveries of machinery regulating vesicle traffic, a major transport system in our cells.

Question 13

What are the different modes of protein transport?

Answer 13

• Gated transport
• Protein translocation
• Vesicular transport

Question 14

What is gated transport?

Answer 14

import into and export out of the nucleus.

Question 15

What is protein translocation?

Answer 15

Protein import into the endoplasmic reticulum

Question 16

What is Vesicular transport?

Answer 16

secretion along the organelles of the secretory pathway

Question 17

What do proteins that don’t reside in the cytosol need in order for them to reach their location?

Answer 17

They need sorting signals.

Question 18

Are sorting signals part of a protein’s sequence?

Answer 18

Yes

Question 19

What can sorting signals be?

Answer 19

• 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.

Question 20

What happens to sorting signals?

Answer 20

• 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

Question 21

How do proteins and other macromolecules (e.g. ribosomes) move between the cytoplasm and nucleus (in and out)?

Answer 21

Via large aqueous nuclear pore complexes (NPC).

Question 22

How many nuclear pore complexes does a mammalian nuclear pore complex (NPC) contain?

Answer 22

A mammalian nuclear envelope contains 3000-4000 nuclear pore complexes (NPCs)

Question 23

Describe the structure of a nuclear pore complex

Answer 23

• 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.

Question 24

How large are ribosomes?

Answer 24

3,300kDa

Question 25

How large are nucleoporins?

Answer 25

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

Question 26

'Small molecules diffuse extremely fast (almost free flow) through Nuclear Pore Complexes'. Is this true?

Answer 26

Yes

Question 27

What would a small molecule be classed as?

Answer 27

5 kDa or less

Question 28

How quickly do proteins of 20-40 kDa diffuse?

Answer 28

They diffuse a lot more slowly

Question 29

Can proteins larger than 40kDa diffuse freely through a nuclear pore complex?

Answer 29

No, and instead, they move through active transport.

Question 30

What do proteins that are larger than 40kDa carry?

Answer 30

They carry nuclear localisation or export signals.

Question 31

What is the diffusion barrier caused by?

Answer 31

The diffusion barrier is caused by unstructured (disordered) regions of channel nucleoporins rich phenylalanine-glycine (FG) repeats forming a tangled network (mesh, or matrix), blocki ng the passive diffusion of large molecules (e.g. ribosomes).

Question 32

Describe nuclear localisation signals (NLS)

Answer 32

- 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).

Question 33

How do FG repeats facilitate active transport through the Nuclear Pore Complex (NPC) ?

Answer 33

- 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

Question 34

Gated transport – ACTIVE nuclear import ACTIVE how?

Answer 34

- 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

Question 35

What does the energy for the active transport come from?

Answer 35

The energy for this type of transport comes from GTP via the GTPase-Ran

Question 36

The GTPase Ran..

Answer 36

- GTPase ‘switch’: - Hydrolyses GTP to GDP (binds both) - Conformation is different depending if bound to GTP or GDP - different conformation = different activity

Question 37

In the nucleus: RanGTP binds importins, causing them to release cargo.

Answer 37

In the nucleus: RanGTP binds importins, causing them to release cargo.

Question 38

'Ran-GTPase provides both the free energy and the directionality for nuclear transport'. Is this true?

Answer 38

Yes

Question 39

What is the net import of cargo driven by?

Answer 39

Net import of cargo is driven by two conformational states of the GTPase Ran, that depend on whether Ran binds GDP or GTP

Question 40

Ran-GDP...

Answer 40

Ran-GDP has conformation (A) and is in this state only in the cytosol

Question 41

Ran-GTP...

Answer 41

Ran-GTP has conformation (B) and is in this state only in the nucleus

Question 42

What is Ran-GTPase regulated by?

Answer 42

Ran-GTPase is regulated by GAP and GEF

Question 43

What is GAP?

Answer 43

GTPase activating protein (promotes hydrolysis of GTP to GDP)

Question 44

What is GEF?

Answer 44

guanine nucleotide exchange factor (exchanges GDP with GTP)

Question 45

Nuclear import (ACTIVE TRANSPORT): summary

Answer 45

- 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

Question 46

Unlike other signal peptides for protein sorting, the NLS is not cleaved after import because:

Answer 46

- 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

Question 47

Is it true that nuclear export is like nuclear import, but in reverse?

Answer 47

Yes

Question 48

What are Nuclear export signals (NES) 'seen' by?

Answer 48

Nuclear export signals (NES) are “seen” by soluble nuclear export receptors - exportins.

Question 49

Exportins...

Answer 49

- 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)

Question 50

How can we visualise subcellular organelles in vivo?

Answer 50

We can visualise subcellular organelles in vivo using dyes or Fluorescent Proteins such as GFP.

Question 51

Is nucleo-cytoplasmic trafficking reversible?

Answer 51

Yes

Question 52

What are other roles of NPCs?

Answer 52

- 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

Question 53

SUMMARY

Answer 53

- 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

Question 54

Is it true that proteins are made in the cytosol, but function in various compartments?

Answer 54

Yes

Question 55

Is it true that sorting signals are like built-in tags that guide proteins to their correct destination?

Answer 55

Yes

Question 56

Is it true that proteins are made in the cytosol, but function in various compartments?

Answer 56

Yes

Question 57

How do proteins enter and exit the nucleus?

Answer 57

Via the nuclear pore complexes

Question 58

Is is true that the Nuclear Localisation Signals for nuclear import can be anywhere on the protein, but must be exposed?

Answer 58

Yes

Question 59

Where is Ran-GTP located?

Answer 59

Inside the nucleus

Question 60

Where is Ran-GDP located?

Answer 60

Inside the cytosol

Question 61

Summarise the steps of nuclear import

Answer 61

- Protein with NLS binds to importin - Importin carries protein into nucleus via NPC - Inside nucleus, Ran-GTP binds importin, releasing cargo - Importin–Ran-GTP exits nucleus - In cytosol, Ran-GAP hydrolyses GTP to GDP, resetting importin

Question 62

Summarise nuclear export

Answer 62

- Proteins with nuclear export signals (NES) bind exportins - Exportin binds Ran-GTP + cargo, moves out - In cytosol, Ran-GAP hydrolyses GTP, releases cargo