L6: Nuclear transport, import and export

Why is DNA kept in the nucleus

1. Protect from harmful molsules→ e.g ROS from the mitochdonria

2. Protect from foreign DNA (virus etc)

   • if the cells knows that their own DNA is in the nucleus

   • when there is foreign DNA in the cytoplasm

   • can be sure and be harsh with the foreign DNA

   • to get rid of it

3. Extra regulation

   • gives a gate for transiption factors

   • TFs can be in nucleus or outside nucelus

   • do not need to be constantly brokendown/made again

   • if it is regulated by the entry into the nucleus

Nuclear disassembly and re-assembly during mitosis: in higher eukaryotic cells, when does nuclear disassembly take place

• in prophase of mitosis

• following complete chromosome replication in the previous S phase

Nuclear disassembly and re-assembly during mitosis: Process of nuclear disassembly

1. Increased protein phosphorylation → by cyclin B-CDK1

2. result in nuclear envelope breakdown

3. lamna repolymerises into

   • soluble lamin A/C

   • membrane assoaciate lamin B

4. Nuclear pore complexes (NPCs) disassemble into soluble nucleoporin subcomplexes

5. nuclear membranes fragment into vescicles or tubes

6. chromatin condenses into separated chromosomes until metaphase

   • associates with spindle apparatus

during this period: chromatin is directly accessible to the cytoplasm

Nuclear disassembly and re-assembly during mitosis: following inactivation of CDK-complexes and a sharp fall in kinase activity in anaphase…

• lamins and NPC proteins become dephosphorylated

→ causes nuclei re-assembly

What does this cause: Nuclei re-assembly

Reassemble in telophase after successful sister chromatid separation

→ inversion of the disassembly process involving:

1. chromosome decondensation

2. membrane assembly from vescicles

3. lamina polymerisation

4. NPC assembly from soluble nucleoporin subcomplexes

But now the nuclei have reassembled→ what must happen next?

• several million proteins and RNA molecules must travel across the nuclear envelope per minute during interphase

• All mediated by nuclear pore complexes…how?

Transport acrosss the nucler envelope

• nuclear portins

• highly conserves

• rigid strucutre→ opens up   gate

• Passive diffusion→ only with smaller than 9nm or 60kDa

  • Everything bigger needs energy and help

Nuclear pore complexes: strucutre (from EM)

• ring of 8 subunits surrounding a central channel 

  • through which proteins and RNA pass

  • with cytoplasmic filamers→ collapses from the cytosplasm>

  • 8 subunits of NULCEOPORINS

    • subunits have different proteins themselves

• fibrils project from both surfaces of the nuclear pore complex

  • those on the inside are organised as basket

  • Well maintained (also called a cage)

What occupies the central channel (gate) through the NPC

The nuclear pore is 40nm BUT only 9nm can diffuse through→ MUST be some kind of gate in the centre:

Protein structure lining and occupying the central gate of the NPC:

• consists of nucleoporin proteins

  • very rich in repeats f the 2 hydrophobic amino acids phenylalanine and glycine (FG)

  • FG repeats can interact with each other and form a dynamic hydrogel

    • there are several models postulated about this strucutre

OUTER→ has a central gate n the middle→ somehthing blocking entry/exit

INNER→ shows the rigid opens in the inner side of the basket

What is the gate?

• selectivity barrier for macromolecules

→ assembled from nucleoporin proteins containing extended domains with FG repeats

→ have FG (phenylalinine and glycine) rich repeats

• forms hydrophobic (blue blobs) along the protein

• at irregular intervals

Next question to ask

• how can interaction between the translocating proteins 

• and the F/G rich repeats favilitate NPC passage

Several models of how synamic hydrogel forms: Virtual gate 

Several models of how synamic hydrogel forms: how does virtual gate model work?

• green→ macromolecules

• red→ translocating molecules, interacting with FG repeats

RESULT: does not show selective transport

CANNOT BE THE CORRECT MODEL

Several models of how synamic hydrogel forms: how might the hydrophobic blobs interact

• formation of a hydrogel

→ leads more to the selective phase model

Several models of how synamic hydrogel forms: how the selective phase model

• red→ blocks pore

• green→ no selectivty

RESULT: selective importation

Several models of how synamic hydrogel forms: experimental evidence for this

• forming the Nsp1 repeat domain

• if mutated protein→ no longer a gel!

RESULT: must be true

Several models of how synamic hydrogel forms: what phase model is favoured

SELECTIVE PHASE MODEL:

• hydrogel of cohesive FG-repeat proteins

  • form a barrier against diffusion of general marcomolecules 

  • whilst providing a solvent for translocating molecules

Size limit to rapid entry:

• prevents entry of larger molecules

  • e.g labelled dextrans of various sizes

• allows passive diffusion of small molecules

Therefore effective pore diameter is…

9nm

effective pore diamter vs channel of the pore complex

• effective pore diameter (9nm) is much smaller than the 60nm channel of the pore complex 

• effective pore diamtere is controlled by the physical properties of the hydrogel

→ TOO SMALL for proteins larger than 40-60 kDa

If it is too small for proteins larger than 40-60kDa, than we have a problem….

• how do large RNA molecules and large proteins interact with the hydrogel and cross NPC

Assays to study import

Using frog oocytes

1. pentamer protein from a frog with radioactive nucleoplasmin microinjected→ allowe in

2. Need to check what is actually causing the importation→ take off the radioactive tails→ no uptake

3. Just microinject the tails→ UPTAKE

but is this actually through the pores??

4. Add tails to colloidal gold particle→ (attached to something that isn’t a protein

   • RESULT: there is uptake into nucleus through nuclear pores

Tail coated gold particles

• Gold particles all line up and enter through nuclear pore

→ gold is too big a particle→ so highlights where the pores are

Import of nuclear proteins: what proteins can accumulate in the nucleus

• proteins of all sizes can accumulate in the nucleus by >100-fold

Import of nuclear proteins: in microinjection experiments into the cytoplasm

• nuclear proteins re-accumulate in the nucleus

how is this acheived:

• selective entry through the nuclear pore complexes

Import of nuclear proteins: underlying molecular mechanism has been eluciated by…

Studying the import of the nuclear proteins:

1. nucleoplasmin

2. SV40 large T antigen

Nuclear localisation signals (NLS)

1. proteins localisaing to the nucleus

   • e.g nucleoplasmin and SV40 large T antigen

2. contain small peptide motifs that cause their import

3. when fused to other non-nuclear proteins (E.g BSA)→ cause nuclear accumulation of the fusion protein

   • bind to specific carrier proteins terms importins (karyopherin in yeast)

• highly conserves and can be transferred into other things→ causes ther things to be transported across

• But can be species specific→ e.g bird flue cannot be transported as well across the human nucleus as human flu can be

Forms INTEGRAL part of the protein

Why important to have an NLS

1. ensures that it is recognised for import VIA proteins

2. ensure that the whole protein doesn’t have to be a specific property etc to get through the hydogel

3. instead→ it is the import that does the work and so the protein only needs a TINY NLS to allow it to be able to get through the nucleus

→ otherwise→ only a limited range of protteins would be able to get through the gel

NLS modifications

• Forms an integral part of the protein

BUT

• can be masked → so can stop/start its import

• Can also have BOTH NLS and Nucleuar Export signals

• ca change masking on each to swtich on and off (in/out)

What do mutations of these proteins cause

• mutation to lysines of nucleoplasmin

• mutation of threonine or asparagine SV40 T antigen 

→ abolish transport

The bold amino acids of the respective NLS were found to be essential”

What did injection of colloidal gold coated with nucleoplasmin into the cytoplasm reveal

• passage through the central channel of the NPC

Nuclear transport: in two steps

1. rapid binding of the cargo to the cytoplasmic side of the nuclear pores

2. then, slower energy-dependent translocation through pores

What is required for these two steps

soluble key proteins:

1. Importin 

2. Ran→ a small GTP-ase

What is the process with these proteins

1. alpha subunit of importin binds the NLS of the cargo

2. the beta subunit docks at the nuclear pore complex

   • importins have spots that are hydrophobic and stick to the hydrophobic parts of the net

   • allows the net to open up as it moves in

   • but close again as it moves through

3. following transport through the NPC, nuclear ran-GTP causes dissociation of importin ffrom NLS-cargo

4. Importin alpha and beta are separately exported back into the cytoplasm in a ran-GTP dependent manner

importin beta reversibly interacts with FG repeats of the NPC

Evidence for the importance of all three features

1. Nuclear localisation signal

2. importin

3. ran

Roles of importin subunits

1. Alpha→ binding to the NLS of cargo

   • import receptor for many cargoes

   • binds to importin-beta

2. Beta→ binding to NPC and ran

   • interacts with FG repeats

   • is the actual import mediator

   • bigger than alpha

   • can bind to hydrophobic interaactions (see previously)

Importin beta reversibly interacts with FG repeats of the NPC → shows the hydrophobic interaction between the blobs and the beta subunits

THEREFORE: it can cross the hydrogel and act as import mediator through the gate

Experimental evidence for diffusion of a transport receptor into hydrogel (seen above: experiment set up

• chamber with buffer

• uses phase contrast and fluorescent microscopy

Measures the diffusion into FG-hydrogel of:

1. A-IBB-RedStar (140kDa)→ importin beta binding domain of importin alpha

2. B-IBB-Redstar in complex with importin beta (530 kDa)

Experimental evidence for diffusion of a transport receptor into hydrogel (seen above: results

Left: buffer

Right: hydrogel

1. with IBB-RedStar→ NO protein transfer

2. with IBB-RedStar/ WITH importin beta complex→ HAD SOME IMPORTED protein

The ran cycle: small GTP’ase ran is involved in the nuclear import (and export)

1. ran can bind GDT or GTP

   • Ran GDP is predominantly cytosolic

   • ran GTP is predominantly nuclear

2. in the cyotsol, ran-sepficic GAP (GTPase activating protein) stimulates the endogenous GTPase of ran

   • e.g RanBP1/2 and RanGAP

3. converts it to the GDP form

4. in the nucleus, chromatin-bound nucleotide exchnage factor RCC1 promotes exchange of the bound GDT to GTP (Ran GAF?)

   • RCC1 also regulates chromatin condensation

5. thus building a ran-GDP/ran-GTP gradient across the nuclear envelope

Ran GDP vs Ran GTP

1. Ran GDP→ cytosolic

   • required for cargo binding to importins in the cytosol

2. Ran GTP→ nuclear

   • promotes cargo dissociation from importins in the nucleus

   • conversely, ran GTP is involved in cargo binding to exportins in the nucleus

Asymmetric distribution

how to remember→ C is before N in the alphabet and D is before T in the alpha bet→ therefore cytosol has more Ran GDP

Regulated import: what is required for the regulation of nuclear import of NLS-containing cargo

• ran cycle be coupled to the importin cycle

Summary of the regulated import/export

note: with the import, there is then export of the proteins that help this import in→forms an equilibirium of masses in and out

1. NLS recognised by alpha subunit of importin and binds

2. beta subunit then binds using GTP

3. interact and bind to the pore

4. moves it through the pore

5. beta subunit has higher affinity for RanGTP which high in the nucelus

6. beta subunit dissociate off and is stable with the RanGTP

7. alpha subunit then off laods

Next the alpha and beta subunits both are exported back into cytoplasm for further import:

alpha subunit:

1. CAS binds and then ranGTP binds

2. goes through pore

3. in cytosol, RanGTP-CAS is unstable and dissociates off with hydrolysis

4. form Cas + Ran GDP

beta subunit

1. stable as beta-RanGTP 

2. moves through pore

3. now unstable and ranGTP hydrolsyses off to RanGDP

4. beta subunit now free for further cargo

note: all of the hydrolysis and removal of RanGTP from subunits also uses the RanBP1 and RanGAP for activation (seen above)

Export of proteins and RNA

• RNA is exported as protein complexes

• signal dependent and carrier mediated

• occurs through the nuclear pore complex

What is the process of this export of proteins and RNA

1. giant mRNA transcripts from insect salivary glands (35-40kb) unfold to pass through the pore

2. always 5’ first

3. before mRNA is exported→ must be processed correctly

   • including splicing and poly A addition

Nuclear export signals (NES)

• have been recently been identified in several exported proteins

• including some that bind RNA

How is export regulated

• export signals are recognised by export receptors (exportins) that are regulated to importin beta

• the regulation of cargo export again depends on the ranGTP/ranGDP gradient across the nuclear envelope

• ran protein levels are equilibrated across the evelope by the nuclear transport factor NTF2

  • ensures there is enough on either side for import/export

Process of export regulation

1. HV unspliced transcipts bind to the Rev protein

2. this mediates export via interaction with an exportin

3. Small RNAs bind their own exportins directly

   1. tRNAs interact with exportin-t

   2. other small non-coding RNAs such as microRNA precursosrs interact with exportin 5

Higher levels of regulated transport (ensure import/export only happen when you want it to): 1 some proteins…

• can only enter the nucleus when released from cytoplasmic anchors

  • e.g: several transciption factors

  • cytoskeleton

  • plasma membrane?

• → this provides a possible level of gene regulation

Higher levels of regulated transport: 2 Phosphorylation of NLS or NES eleents

another discussed means to regulate nuclear transport:

e.g:

1. cyclin B1 constantly shuttles between nucleus and cytoplasm in S and G2 phase

2. but phosphorylation of NES is early mitosis BLOCKS export

3. → results in nuclear accumulation

See again: regulation model 2 (involving protein phsophrylation)

Regulation model 2b (involving protein phosphorylation)

Regulation model binary switch

• when the protein has BOTH NLS and NES

• can be masked on and off

  • or may be both on

• Regulates the ferrying between nucleus and cytosplasm