Moving into the nucleus

Question 1

What separates eukaryotes from prokaryotes?

Answer 1

The ability for the cell to compartmentalise distinct processes within organelles.
A cell is eukaryotic if it has membrane-bound nucleus and other organelles enclosed in a membrane.

Question 2

How is modulating interactions by localistion to membrane bound compartments important?

Answer 2

Localisation enables the cell to modulate interactions between proteins.
This increases the relative concentrations of interacting species, more likely to see interaction or catalysis occurring.

Question 3

How is regulation by localisation to membrane-bound compartments important?

Answer 3

Allows regulation of the local environment.
Transport of vesicles by motor proteins along the cytoskeleton, enabling regulation within distinct cellular domains.
e.g. Gene expression happens in nucleus of cell body, but proteins need to move into synapse for their function

Question 4

How is a distinct environment by localisation to membrane-bound compartments important?

Answer 4

Provides a distinct environment allowing for specialised functions.
Lysosomes have a low pH for protein degradation.

Question 5

How is protection by localisation to membrane-bound compartments important?

Answer 5

Protects the microenvironment from toxic metabolites.
e.g the mitochondria produces Reactive oxygen species, if not immediately converted to H2O2 by detoxifying enzymes it is dangerous.
By compartmentalising ROS cannot disrupt other cellular processes.

Question 6

How is material exchanged between organelles?

Answer 6

Gated transport
Transmembrane transport
Vesicular transport

Question 7

What is gated transport?

Answer 7

Occurs through pore complexes
Bidirectional between cytoplasm and nuclear pore complex

Question 8

What is transmembrane transport?

Answer 8

Involves direct passage across intact membranes.
E.g. a diffusion gradient for small molecules.
Proteins usually require unfolding.
Happens between mitochondria, ER, Plastids, and Peroxisiomes between the cytosol

Question 9

What is vesicular transport?

Answer 9

Does not involve passage across the membranes - involves fusion of membranes.
Exocytosis and endocytosis.
Secretory vesicles, linked to proteins having an N-terminal signal peptide - ER and Golgi transport

Question 10

What is Swyer Syndrome?

Answer 10

SRY mutation, expressed on Y chromosome.
Mutation causes no expression of testosterone and expression of female gentalia, despite being genetically male (XY).

Question 11

What does compartmentalisation of the nucleus do?

Answer 11

Separates genetic material from the rest of the cell.
Uncouples gene transcription from protein synthesis.
So the processes are highly regulated.
But the nucleus must communicate with the rest of the cell to enable gene expression.

Question 12

What is the nuclear membrane?

Answer 12

A phospholipid bilayer.
Outer membrane is continous with ER.
Inner membrane has underlying meshwork of filaments - nuclear lamina.

Question 13

What is the nuclear lamina?

Answer 13

A critical nuclear scaffold.
Creates and maintains structure intergrity.
Comprised of lamin (intermediate filaments), studded with lamin associated proteins (LAPs)

Question 14

What is Lamin A?

Answer 14

Processing of mature Lamin A from pre-Lamin A is complex.
Farnesylation allows for rapid delivery of Lamin A to the nuclear envelope.

Question 15

What is lamina for?

Answer 15

Gene regulation
NPC assembly
Chromatin function
Cell division
DNA replication

Question 16

What do mutations in Lamin A do?

Answer 16

Causes premature aging syndrome Progeria.
Don’t get cleavage of C-terminal, causes changes in shape in nuclear architect and when cell divides - DNA damage.

Question 17

What is the Nuclear Pore Complex?

Answer 17

NPC allows free movement of small molecules and selective transport of larger proteins - molecular sieve.
Nuclear pores are dismantled during mitosis and rebuilt after every cell cycle.

Question 18

What is the NPC structure?

Answer 18

500 subunits of 30 nucleoporins (Nups).
Assembled in 8-fold symmetric structure that studs the double-nuclear membrane together.
Centre structure encases the transport channel.
Peripheral structures connect NPC to local environment.

Question 19

What are the types of Nups?

Answer 19

Outer ring
Transmembrane
Inner ring - these 3 anchor NPC to nuclear membrane
FG Nups - unstructured, forms a hydrogel sieve.
Held in place by Linker Nups to centre of pore.

Question 20

What are FG Nups?

Answer 20

Contain FG motifs - phenylalanine-glycine.
Interjected by spacer amino acids, allow for flexibililty.
Phenylalanine is aromatic, can form interactions with neighbouring phenylalanine.
Enables passage of proteins.

Question 21

Why is the aromatic ring in FG nups important for hydrogel?

Answer 21

Mutation to a non-aromatic ring disrupts hydrogel barrier - nuclear pore becomes more porous.
Mutation to another amino acid with an aromatic ring preserves the hydrogel barrier, Van Der Vaals can still form.

Question 22

How does movement occur within NPC?

Answer 22

Some by passive diffusion - metabolites, ions and small proteins.
Most by energy dependent transport - proteins, mRNA, tRNA, ribosomal subunits, viruses.

Question 23

How is the import and export of proteins regulated?

Answer 23

The transport is a cycle between protein cargo, which interacts with nuclear transport receptors importins and exportins, and small GTPase Ran.

Question 24

What is the process of import/export of proteins across the nuclear membrane?

Answer 24

Cargo is recognised and bound by NTRs.
The NTR-cargo docks on the NPC via FGnups. Cargo is translocated through the NPC.
The NTR-cargo is dissociated.

Question 25

How do proteins target the nucleus?

Answer 25

Nuclear Localisation sequence - a run of 5 basic amino acids - directs cytosolic proteins to the nucleus.
Can be anywhere in a protein sequence.

Question 26

How are NLS mapped?

Answer 26

STAT3 fused to GFP.
Localisation is revealed by GFP fluorescence.
Small chunks of amino acid can be removed and shows change in localisation.

Question 27

What are Importins?

Answer 27

Or Karyophilins:
Heterodimer of importin-a and B.
Alpha recognises NLS on cargo protein.
Beta interacts with the pore complex via FG Nups within the NPC.

Question 28

What is movement through the pore?

Answer 28

Movement is random, but directionality of cargo import is determined by RanGTP gradient.
RanGTP binds importin B and causes release of the cargo.
RanGTP levels are higher in the nucleus than cytoplasm.

Question 29

What is the Ran GTP-GDP gradient?

Answer 29

Ran-GDP is cytosolic.
Ran-GTP is nuclear.
High levels of Ran GTP enables dissociation of cargo from beta importin.
Enables movement into nucleus, and recycling of importins back to cytoplasm.

Question 30

How is the Ran GTP/GDP gradient maintained?

Answer 30

Ran-GEF enzyme loads GTP to Ran, localised to chromatin.
Ran-GAP hydrolyses Ran GTP to GDP, localised on cytoplasmic filaments of NPC.

Question 31

What is phosphorylation of cargo?

Answer 31

Mainly increases import - increased binding of cargo to importin-a, enhanced recognition of cargo by alpha, and induced conformational change which exposes NLS.
Can also decrease import by inducing cytoplasmic retention.

Question 32

How does phosphorylation increase binding affinity of cargo to importin-A?

Answer 32

EBNA-1 protein, important for replication of DNA of EBV virus.
Phosphorylation at Serine increases affinity, if mutated EBNA cannot enter nucleus, so shows phosphorylation is important.

Question 33

How does phosphorylation enhance recognition of cargo by importin-a?

Answer 33

SV40 large T antigen.
Phosphorylation sites upstream of NLS, recognition of NLS increased 100 fold.

Question 34

How does conformational change by phosphorylation work?

Answer 34

In STAT1 transcription factor, the phosphorylation induces dimerisation, this conformational change exposes the NLS.

Question 35

How does phosphorylation induce retention?

Answer 35

NFAT transcription factor is a mediator of T cell activation.
Phosphorylation sites overlap NLS.
High calcium levels activates phosphatase calcineurin, reveals NLS, so protein can translocate to nucleus.

Question 36

How does export of proteins to the cytoplasm occur?

Answer 36

Exportins Crm1 and NXF1, move proteins out.
Nuclear Export Sequences (NES) are leucine rich.
Exportins require Ran-GTP for interaction with cargo, reverse of importins.

Question 37

What is nuclear export of mRNA?

Answer 37

NXF1 recognises 3’ cap on mRNA.
Binds to TRP at NPC basket, enables localisation of mRNA to nuclear pore.
RanGTP gradient is not used for mRNA, instead localisation of GLE1 and DDX19 proteins.
NXF1 is then recycled back to nucleus via importins.