18-22: Pool

Question 1

How does nuclear-cytoplasmic transport fundamentally differ from other protein translocation systems in the cell?

Answer 1

• Substrates can be large and complex
• Cargo is FOLDED
• Bi-directional transport

Question 2

What are NPCs and the key facts about them?

Answer 2

Nuclear Pore Complexes - large structures (125 Mda) with 8-fold symmetry, through which nuclear transport occurs

They are composed of 30+ Nuceloporins (which often contain “FG Repeats” - short clusters of hydrophobic Phe-X-Phe-Gly resides separated by hydrophilic linkers)

There are around 3000-4000 per nucleus

Question 3

What does the proportion of different proteins (e.g., Lysozyme, Ovalbumin, Globulin) that enter the nucleus reveal about nuclear transport?

Answer 3

For small proteins (e.g., Lysozyme, Cyt C), 12% ends up in the nucleus after 24 hours - since the nucleus is around 12% of the cell volume, this suggests small molecules simply diffuse in

Proteins larger than around 60 kDa (e.g., Globulin) cannot diffuse in, as less than 1% ends up in the nucleus

Question 4

Cite and describe the experiment that demonstrated that nuclear transport of larger proteins is selective and requires a signal

Answer 4

Laskey, R. (1982) Nucleoplasmin

When the nucleoplasmin pentamer is placed in the cytosol, it is transported into the nucleus (despite being very large)

When the tail alone was placed in the cytosol, it was transported in

When the core alone was placed in the cytosol, it was NOT transported in

When the core alone was placed in the nucleus, it remained in the nucleus

-> These findings suggest that nucleoplasmin accumulates in the nucleus via Selective Entry and remains in the nucleus via Selective Retention, due to an import signal in the tail

Question 5

Describe the structure and significance of NLSs

Answer 5

Nuclear Localisation Signals:
1-2 stretches of BASIC residues (lots of K and R) which are both necessary and sufficient for nuclear accumulation

Mutants in NLSs block nuclear entry (e.g., SV40 Large T-antigen K128A/T), while the addition of an NLS to a cytosolic protein leads to nuclear accumulation (e.g., Pyruvate Kinase)

In nucleoplasmin, the signal sequence was discovered by repeating the earlier experiment with smaller and smaller tail fragments, until the exact signal region was found

Question 6

What experimental evidence initially implied the existence of NLS receptors?

Answer 6

Adding NLS-albumin conjugates was shown to block the import of most proteins
-> Therefore, the import process is saturatable due to some limiting factor (presumably NLS receptors)

Question 7

What proved that nuclear import requires Cytosolic Factors?

Answer 7

Adam and Gerace (1990)

SV40 successfully imported in control cell

When cell was semi-permebilised with low concentration of detergent, NO import (SV40 remains in cytosol)

When cell was semi-permeabilised, then cytosol + energy added back in, SUCCESSFUL transport

Question 8

After proving that cytosolic factors are required for import, how were these cytosolic factors identified?

Answer 8

Fractioning of the cytosolic extract via gel filtration chromatography

Then, each fraction was tested (as before) with the semi-permeabilised cell import assay

Certain fractions (A and B) allowed some import activity (about half of normal import activity)

Pooling fractions A and B restored almost normal import

Therefore, the key factors must be contained within fractions A and B

Question 9

What are the four soluble factors, required for import of NLS-containing cargo, that were discovered from the fraction assay?

Answer 9

Discovered in Fraction A:
- Importin-alpha (NLS-binding protein)
- Importin-ß (Dimerises with Imp-a and interacts with FG repeats of NPC)

Discovered in Fraction B:
- Ran (small GTPase)
NTF2 (interacts with Ran)

Question 10

What are the two steps of nuclear import (and what experimental evidence demonstrates which fractions are necessary for each step)?

Answer 10

1. DOCKING (energy independent)
   -> Impß interacts with FG sequences of cytosolic fibrils
2. TRANSLOCATION (energy, Ran, and NTF2-dependent)
   -> Cargo carried into nucleus

If JUST Fraction A added, rim staining at Nuclear Envelope (presumably, factors in this fraction dock with NPCs, but no nuclear transport

If add A+B, nuclear staining shows successful nuclear transport (presumably, something in fraction is required for the actual import)

Question 11

Describe the Ran GTPase Cycle

Answer 11

Ran is a GTPase, and is converted from its inactive form (RanGDP) to its active form (RanGTP) by the GEF RCC1

It is inactivated via hydrolysis by RanGAP1

Since RCC1 is mainly found in the nucleus (tightly bound to chromatin) and RanGAP1 is mainly found in the cytosol (excluded from nucleus), Ran is mainly active in the nucleus and inactive in the cytosol

-> A GRADIENT of RanGTP exists across the Nuclear Envelope

Question 12

Describe the experimental evidence for the role of the RanGTP gradient in NLS-cargo import

Answer 12

Use cells expressing a temperature-sensitive mutant of RCC1 (inactive at 39C)

At permissive temperature, GFP-NLS shows successful import into nucleus

At 39C (Western Blot shows RCC1 successfully blocked), GFP-NLS shows lack of nuclear import

Question 13

Describe how the RanGTP gradient affects Importins

Answer 13

Importins bind cargo in the absence of RanGTP (in the cytosol) and carry it into the nucleus

Once inside the nucleus, RanGTP binds to Impß, causing Cargo+Imp-a to be released (which then dissociate from each other)

Impß, bound to RanGTP, is then transported OUT into the cytosol (via the NPCs due to its FG sequence), where RanGTP is hydrolysed to RanGDP by RanGAP1

Impß is then released from the inactive RanGDP, and can bind to Imp-a + Cargo once again

Question 14

Describe the experimental evidence for the Existence of the RanGTP gradient

Answer 14

Use a RanGTP-sensitive probe, and FRET/CFP analysis:

Nucleus appears Blue (high RanGTP) while cytosol appears green (low RanGTP)

Question 15

Given that Imp-a LACKS FG sequences (unlike Impß), how does Imp-a get back into the cytosol (and what is the experimental evidence for this process)?

Answer 15

Nuclear Export - this requires the RanGTP gradient AND Exportins (e.g., CAS)

RanGTP Gradient:
- Experiment on Xenopus oocytes
- Add RanGAP1 to nucleus, so RanGDP in both nucleus and cytosol (no gradient)
- U1 and U5 snRNA fail to export into cytosol (whereas in control cell they are mostly exported)

Exportins:
- Export receptors work similarly to importins, but in reverse
- In the nucleus, Exportins bind RanGTP, which promotes cargo binding, then pass through NPCs to the cytosol
- In the cytosol, RanGTP is hydrolysed to RanGDP, which then dissociates from the exportin, so the cargo is released, and the exportin is transported back to the nucleus

In this case, Imp-a specifically is exported by CAS

Question 16

What is an NES?

Answer 16

A Nuclear Export Signal:
- A short, hydrophobic, Leu-rich sequence
- Found on proteins exported from the nucleus
- Recognised by the export receptor CRM1

Question 17

How can NES-containing proteins play a role in RNA export?

Answer 17

They can act as adaptor proteins - for example:

HIV-1 Rev Protein binds a stem-loop structure in viral RNA, while also binding CRM1 via its NES

Thus, the virus is able to “hijack” the export machinery to transport its genomic RNA into the cytoplasm for packaging

Question 18

What is Exportin-t

Answer 18

The export receptor specifically for tRNA

Question 19

How does Bulk mRNA Transport differ from export of HIV RNA or Importin-alpha?

Answer 19

It does NOT require an Imp-ß like export receptor - instead, splicing and export are coupled via a mediating protein called TAP/Mex67p

This protein forms a complex with Mtr2, allowing it to interact with the NPC

Meanwhile, it does NOT bind the mRNA directly, but rather binds the Exon-Junction Complex of RNA that has been spliced (preventing export of UNspliced RNA)

The requirement for TAP (and the difference in mechanism from nuclear IMPORT) is shown by the mex67-5 ts mutant, which blcoks polyA mRNA export, but does not affect nuclear import

[HIV viral RNA needs an additional adaptor (Rev) precisely BECAUSE it is not yet spliced]

Question 20

Given that each nuclear transport event removes one Ran molecule from the nucleus, how does Ran get back in?

Answer 20

NTF2 (a 10kDa homodimer) binds both RanGDP and NPCs, then carries RanGDP into the nucleus

RanGDP is then converted to active RanGTP by RCC1, and dissociates, allowing NTF2 to pass BACK into the cytosol

Question 21

Describe the role of Ran in Cell Division

Answer 21

Ran is required for the assembly of the mitotic spindle:
- The chromatin appears to promote formation of the spindle in mitotic extract - this is because RCC1 is bound to the chromatin, so this is where RanGTP is concentrated
- Adding RanGDP or RanGAP blocks spindle formation

Mechanism:
- TPX2 is inactive in most areas of the cell as it is bound to Imp-a and Imp-ß
- However, near the chromatin, RanGTP binds Impß, so TPX2 is released and becomes active
- TPX2 then promotes spindle assembly via nucleation and phosphorylation of several proteins
- It forms a complex with NEDD1 and RHAMM to nucleate MTs
- Also activates AuroraA kinase

Conclusion: Ran is a very versatile molecule! (In both roles, it provides POSITIONAL INFORMATION)

Question 22

What is mRNA Localisation and what are some key examples?

Answer 22

While most mRNAs are distributed uniformly in the cytoplasm, some must be specifically localised before translation:
- ß-actin localised at leading edge of cell
- MAP2 and MBP in Neurons
- Ash1 and Oxa1 in S. cerevisiae

71% of Drosophila embryo mRNAs show some degree of localisation

Question 23

What are the most common PURPOSES of mRNA localisation?

Answer 23

• Asymmetric cell division
• Generating cell polarity
• Co-translational interactions
• Localised cellular responses
• Reduced protein transport
• Viral infection

Question 24

What are the main methods for mRNA Localisation?

Answer 24

1. Active, Directed Transport via Cytoskeleton
2. Random diffusion (via Cytoplasmic Streaming) with trapping at key region of cell
3. Generalised degradation except protected mRNA in target region (fast but wasteful method)

Question 25

State the three broad steps in the active transport method of mRNA Localisation

Answer 25

1. Assembly of transport complex
2. Transport along cytoskeleton
3. Anchoring at destination

Question 26

What is the significance of Ash1 mRNA in terms of mRNA localisation and research?

Answer 26

Ash1 is a transcriptional repressor of HO endonuclease, which restricts mating-type-switching to the mother cell only, which must be transported to the bud tip of the daughter cell

Ash1 was the basis of a genetic screen for mutants which showed HO expression in the daughter cell, the components discovered provided insight into the transport mechanism of Ash1 mRNA

Question 27

What were the relevant genes discovered by the Ash1 mRNA localisation screen (and what are the key functions of their products)?

Answer 27

She1 - Type IV Myosin Motor
She2 - RNA binding protein
She3 - Adaptor Protein
BNI1 - Actin Cytoskeleton Organisation Protein

These roles allowed researchers to imagine a possible mechanism for Ash1 mRNA localisation

Question 28

Describe the mechanism behind Ash1 mRNA localisation in S. cerevisiae

Answer 28

1. Assembly of the Transport Complex
   - Ash1 mRNA contains “zip-codes” (stem-loop structures present in the ORF and 3’ UTR)
   - The RNA-binding protein She2p binds to these zip-code elements
   - NOTE: Removing any of the 4 zip-codes affects targeting, while removing ALL prevents targeting
2. ASH1 Transport
   - After binding the stem-loops, She2p recruits She3p (an adaptor)
   - She3p binds to Myo4p (a plus-end-directed myosin motor protein)
3. ASH1 Anchoring
   - Anchoring of Ash1 in the bud tip of the daughter cell requires both Bni1p (an actin cytoskeleton organisation protein) AND translation of Ash1 mRNA
   - Anchoring is necessary to prevent Ash1 drifting off again due to cytoplasmic streaming

Question 29

Describe how Ash1 mRNA transport can be visualised experimentally, and what was proven using this visualisation

Answer 29

Adding a 5th stem-loop to the Ash1 mRNA, which is recognised by the protein MS2, then fusing GFP to MS2 so that GFP shows where Ash1 mRNA is being localised

Using this model, it was proven that She1/Myo4 is required:
- In the WT cell, the complex forms and is seen to move to the bud tip
- In the She1/Myo4 mutant, the complex forms but remains stuck in the mother cell

Question 30

Give a second example besides Ash1 of an mRNA localised by active transport, and explain the mechanism + how it compares to Bcd and Ash1

Answer 30

OSKAR - localised to the posterior end of the Drosophila oocyte (osk is concentrated at the pole plasm, whereas bicoid is concentrated at the head and thorax)

Zip Codes:
Like Ash1, both Osk and Bcd contain stem-loop zip codes in their 3’-UTR
-> These zip codes are both necessary AND sufficient, as fusing the Osk zip code to LacZ causes posterior localisation, while fusing the Bcd 3’-UTR to Osk causes anterior localiastion

Transport Complex:
- STAUFEN binds the Osk zip code, and recruits many other proteins
- One of the proteins recruited by Staufen is BRUNO (which prevents premature translation of Osk before it has been localised)
- Many other proteins recruited by Staufen are part of the E-J complex

Transport of Osk:
- While Ash1 requires AFs, Osk requires MICROTUBULES! (Injecting Nocodazole prevents localisation, while Gurken mutants show localisation at the cell equator)
- Osk transport requires the Kinesin Heavy Chain, which transports it from the minus-end to the plus-end of microtubules (whereas Bcd is transported by dynein in the opposite direction) - khc/khc mutants show no Osk localisation
- The Exon-Junction complex activates Kinesin I (this ensures that only SPLICED Osk can be localised)

Anchoring:
- When the Osk transport complex reaches the posterior end of the cell, Bruno is released, allowing translation to be activated
- Oskar protein is now anchored by actin-binding proteins

Overall: The principles of localisation for Ash1 and Osk are similar, except that Oskar requires MTs while Ash1 requires AFs

Question 31

State and explain an example of an mRNA that is localised by random diffusion and Trapping

Answer 31

NANOS (Nos)
A posterior determinant in Drosophila oocytes which is synthesised after fertilisation

• Its localisation is not directly linked to the cytoskeleton, but requires correct Osk localisation (in Osk mutants, nos is not localised)
• It is trapped at the posterior end by proteins that recognise sequences in its 3’UTR

Question 32

State and explain an example of an mRNA that is localised by local stabilisation

Answer 32

Hsp83 in Drosophila undergoes rapid, generalised degradation following fertilisation (mediated by Smaug, which binds Hsp83 AND an adenylase complex)

However, at the posterior end, Hsp83 mRNA is protected against degradation

Question 33

Give some examples of how organelle requirements vary by cell type

Answer 33

Hepatocytes have extensive smooth ER for lipid synthesis and detoxification

Brown Adipose Tissue has many large mitochondria and fat droplets to convert chemical energy (fat) into heat, e.g., when emerging from hibernation

Question 34

Give some examples of how cellular organelle requirements can change during development

Answer 34

Activated B Cells (Plasma Cells) need increased RER to produce antibodies

Activated effector T cells need increased mitochondria and free ribosomes, and thus appear grainy

Question 35

Summarise the two “Case Studies” for regulation of organelle size and function

Answer 35

1 - The ER and UPR (UPR is a homeostatic mechanism to regulate ER folding, failure of homeostasis triggers apoptosis)

2 - Mitochondria (size and morphology regulated by fission and fusion)

Question 36

Name some of the proteins required for successful ER protein folding

Answer 36

1. Chaperones (two major families - Hsp70 proteins, e.g., BiP, and Carbohydrate-dependent chaperones)
2. Protein Disulphide Isomerases (e.g., PDI) for S-S bond formation
3. Oligosaccharyl transferase (for N-glycosylation) - found in ER membrane, proteins often glycosylated AS they are transported in

Question 37

How was it experimentally demonstrated that cells have a mechanism to respond to increases in ER folding load?

Answer 37

Treatment with DTT or tunicamycin blocks S-S bond formation and N-glycosylation respectively (which artificially mimics physiological settings in which folding demand exceeds capacity)

However, WT cells still grow normally even when treated with these drugs (showing that there is a cellular response to folding load)

Question 38

Describe the UPR and its mechanism in yeast

Answer 38

Unfolded Protein Response - how the cell responds to increased ER folding load (by increasing transcription of folding machinery)

A screen for yeast mutants that CANNOT grow in DTT/tunicamycin revealed:
- Ire1 (a Kinase/Endonuclease in the ER membrane)
- Rlg1 (an RNA ligase)
- Hac1 (a TF)

Misfolded proteins bind Ire1, oligomerising and activating its Kinase domain -> trans-autophosphorylation activates the Ribonuclease domain, which then splices the Hac1 pre-mRNA

Rlg1 then ligates the Hac1 exons together so that Hac1p can be translated

Upregulated Hac1 then enters the nucleus and induces transcription of ER chaperones, such as Car2p, Cne1p, as well as other proteins which modify or transport proteins to assist the UPR

ER chaperones then bind misfolded proteins in the ER, and promote proper protein folding, e.g., Hsp70s do this by binding exposed hydrophobic domains found on unfolded proteins

Question 39

Describe how the UPR Pathway in higher eukaryotes differs from yeast

Answer 39

Firstly, many components are conserved, and show homology with components in the yeast pathway (e.g., IRE1 -> Ire1; XBP1 -> Hac1; tRNA ligase complex -> Rlg1)

However, system is more complex:
- 3 ER membrane proteins that sense misfolded proteins
- As well as IRE1, there is PERK (similar to Ire1 but lacking ribonuclease activity, instead it phos-inactivates TRANSLATION INITIATION FACTORS to reduce folding stress on ER; and it promotes selective translation of Gene Reg Prot 2
- Also ATF6 (a TF normally tethered to ER membrane, but folding stress causes GRP78 to dissociate from it, allowing ATF6 to be cleaved by S1P and S2P, causing it to translocate to the nucleus and activate protein folding)

Having 3 different pathways allows more fine-tuning, and a range of responses (e.g., only PERK reduces translation to ease the folding load)

Also note: In tissues with a high secretory load, e.g., pancreas, the UPR can be turned on even under normal physiological conditions - in fact, mice lacking PERK develop diabetes due to lacking this mechanism

Question 40

Describe the relationship between UPR and apoptosis

Answer 40

Prolonged, excessive ER folding stress that fails to restore homeostasis can result in apoptosis

A number of pathways for this:
- PERK -> CHOP + NOXA/BIM
- Ire1-alpha also upstream of CHOP

This is fine if only a few cells undergo apoptosis, but in protein misfolding diseases, these pathways can lead to large-scale cell death + disease symptoms

Question 41

What is meant by “Mitochondrial Dynamics”

Answer 41

Cells can change the number AND morphology of mitochondria to adapt to changes in physiological conditions (e.g., exercising mouse has more, bulkier mitochondria)

In cells, mitochondria form a reticulum, or network, which is dynamic due to fission and fusion

Question 42

How are fission and fusion regulated, and why are they important?

Answer 42

Regulated by dedicated and distinct GTPases

Important for inheritance and distribution:

• If a cell didn’t inherit any mitochondria, nor any mDNA -> Fission prevents this
• Without fission, all the mitochondria could cluster together at the cell centre, and fail to serve the cell periphery with energy

Question 43

Which type of cells are most affected by defects in mitochondrial dynamics, and why?

Answer 43

NEURONS
- High energy demand
- Strong reliance on mitochondrial distribution

Defects in mitochondrial dynamics linked to progression of Parkinson’s

Question 44

What are the main purposes of autophagy?

Answer 44

1. Restructuring cells during development
2. Adaptive response to Starvation, Stress or Infection
3. Disposal of Bulk Cytoplasm, Large Protein Aggregates OR Whole Organelles

Question 45

What are the two broad types of autophagy?

Answer 45

1. NON-SELECTIVE
   (Bulk selection of cytoplasm - allows recycling of macromolecules, so C/N skeletons can be used as nutrients)
   - Often triggered by starvation signals
2. SELECTIVE (Specific cell components)
   Mitophagy/Pexophagy/ER-phagy/Ribophagy
   - Triggered by signals on specific organelles

Question 46

Describe how autophagy occurs (steps and components)

Answer 46

1. Initiation (de novo) and extension of phagophores/isolation membranes
2. Closure
   (Pre-autophagic membrane engulfs cytosol and organelles)
3. Transport and fusion with lysosomes
4. Digestion (by lysosomal enzymes)

Question 47

What is the key (mentioned) protein in autophagy and what is its role?

Answer 47

Atg8 (mammalian homologue: LC3) - actually a family of proteins that are involved in several stages of autophagy

Tethered to the pre-autophagic membrane by a covalent lipid anchor
Then recruits various adaptors which act as receptors for different autophagic substrates
ATG8 lipidation is analogous to ubiquitination

Question 48

How does a cell “know” which mitochondria are damaged/non-functional and thus need recycling?

Answer 48

A protein called Pink1 (which plays many roles in mitochondrial homeostasis) is targeted to mitochondria, but in healthy cells is sequentially cleaved and degraded

However, if mitochondria become defective (e.g., due to accumulation of oxidative damage caused by ROS), the inner membrane is less polarised, blocking protein import, therefore Pink1 accumulates on the surface

On the surface, Pink1 is stabilised by TOM proteins, and phosphorylates Mitofusin2 (Mfn2) which then recruits PARKIN (see other FC for more on Parkin)

Question 49

Describe the role of Parkin in mitochondrial homeostasis, and the consequences of mutations in the Parkin gene

Answer 49

Parkin is a ubiquitin E3 ligase

When recruited to the OMM by Mfn2 (downstream of Pink1 and mitochondrial damage), Parkin ubiquitinates multiple mitochondrial membrane proteins, triggering the autophagic machinery

Aut. recessive mutations in Parkin are linked to early onset Parkinson’s, as blockage of mitophagy leads to accumulation of damaged mitochondria, which leads to disease presentation in CNS (as neurons are most severely affected)

Question 50

Briefly describe the structure of ubiquitin

Answer 50

It is a small, conserved protein (76 AAs)

Hydrophobic globular core, and exposed C-terminus (which is the site of attachment to target proteins)

Lys48 is conserved and exposed

Question 51

What happens to misfolded proteins IN THE ER that must be disposed of?

Answer 51

If they are left to accumulate in the ER, this may interfere with folding of other proteins, so they must be removed

Since there is no degradation pathway inside the ER, they must undergo RETRO-TRANSLOCATION into the cytosol, and make use of the degradation machinery for cytosolic proteins

Question 52

Describe the process of ubiquitination

Answer 52

1. ACTIVATION
   - An E1 binds ubiquitin via its C-terminus in a S-C(-Ub)=O bond [This requires ATP -> AMP + 2Pi]
   - An E2 binds to E1-Ub, and the S-C(-Ub)=O group is transferred to the E2, which is now PRIMED with Ub
2. CONJUGATION
   - An E3 (bound to the primed E2) binds to a target protein due to recognition of the degradation signal
   - The first Ub chain is added to the target protein via the NH2 group of a Lys side chain on the target
3. POLYUBIQUITINATION
   - Further Ub chains are added to the first one via K48

Question 53

Describe how Specificity is achieved in Ubiquitin enzymes (in S. cerevisiae)

Answer 53

E1 (Ubiquitin-activating enzyme) - just 1 gene

E2 (Ubiquitin-conjugating enzymes) - 13 different genes

E3 (Ubiquitin-protein ligases) - multiple families, over 100 genes

Question 54

Describe the structure and function of the proteasome

Answer 54

The proteasome is a large protease complex which carries out degradation of ubiquitinated proteins

The 20S core contains rings of seven alpha, seven ß, seven ß and seven alpha subunits

The 19S Caps form a barrier to compartmentalise the active sites, which are found on the ß-subunits, allowing the cell to regulate which proteins reach them, and avoid unnecessary degradation

Specifically, the 19S Cap recognises poly-ubiquitinated proteins and deubiquitinates them (Ub-C-terminal hydrolase); ATP-dependent unfolding of the substrate then allows it to pass through the hexameric ring structure of AAA-ATPases into the 20S core, where it remains bound to the proteasome until it has been fully degraded into short peptides, which are then hydrolysed into individual AAs

Question 55

What happens to large cellular aggregates in the ER or cytoplasm?

Answer 55

These aggregates are too large to undergo retro-translocation + ERAD, or proteasome degradation, respectively

Instead, they are degraded by ER-phagy or Autophagy (involving p62 and Atg8) instead, respectively