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Molecular cellular biology > Liz Smythe lectures - protein sorting and vesicular transport > Flashcards

Liz Smythe lectures - protein sorting and vesicular transport Flashcards

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1
Q

how is material moved in a cell?

A
  • material, signals and nutrients move into cells by endocytosis
  • cells deliver material via the secretory pathway by the ER and Golgi to the cell surface and outside of cell
  • material can also be transported to organelles within the cell
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2
Q

why is compartmentalisation of eukaryotic cells important?

A

it is key for the specialised function of eukaryotes
- transport systems and targeting systems ensure that the right proteins go to the right places in the cell

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3
Q

what are the different transport types within the cell?

A
  1. gated transport between the nucleus and the cytoplasm
  2. transmembrane transport of post-modified proteins from the cytosol to organelles such as the mitochondria, chloroplasts, peroxisomes
  3. vesicular transport is used to move proteins between the organelles of the secretory pathway
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4
Q

what is the common theme to all protein sorting?

A

signals are required for protein sorting

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5
Q

what is the nuclear pore?

A

it allows material to move in and out of the nucleus
- it brings together the double membrane of the nucleus, so that the nucleoplasm can interact with the cytosol
- nuclear pore is formed at the junction of the inner and outer membranes of the nuclear envelope

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6
Q

what is the structure of the nuclear pore complex?

A
  • the complex consists of multiple copies of ~30 different nucleoporins
  • each complex is made of 8 subunits with a central plug
  • nucleoporin dye is seen around the rim of the nucleus, showing that the pores are localised at the nuclear envelope
  • rings of nucleoporins surround the central pore
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7
Q

what is the function of the nuclear pore complex?

A
  • nuclear pores are involved in moving substances across the nuclear envelope between cytosol and nucleus
  • Gating mechanism allowing certain molecules into/out of nucleus
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8
Q

what are examples of the function of the nuclear pore?

A
  1. In the synthesis of DNA, histone molecules are required to package the DNA
    - Histones are made in the cytosol by ribosomes and then are transported from the cytoplasm into the nucleus
  2. For protein production to occur, ribosomes are needed.
    - the ribosomal subunits formed in the nucleolus must enter the cytoplasm via export through the nuclear pore complexes
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9
Q

what are the 2 ways in which substances are transported across the nuclear pore complex?

A
  1. simple diffusion
  2. active diffusion - nuclear translocation requiring energy
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10
Q

how is transport across the nuclear pore size limited?

A

simple diffusion can only occur up until molecules of 60kDa:
- Up to 5000Da = freely diffusible
- 5000-17000Da = 2 minutes to equilibrium
- 17000-44000Da = 30 minutes to equilibrium
- 60000Da = cannot enter by diffusion

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11
Q

how do molecules that are larger than 60kDa enter the nucleus?

A

nuclear translocation - active transport
- molecules larger than 60kDa are excluded from the nucleus unless they provide a signal
- the nuclear translocation signal is needed for these large molecules to interact with the nuclear pore
- under the appropriate signal, the pore can open up to 26nm in diameter

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12
Q

what is the signal that triggers nuclear translocation?

A

In the case of proteins, the signal is linked to a peptide sequence
- Nuclear transport recognition sites are rich in Lys, Arg and Pro
- A mixture of these amino acids is sufficient to transfer protein into nucleus

Example: the T-antigen of the SV40 virus contains the sequence Pro-Pro-Lys-Lys-Lys-Arg-Lys-Val, so can translocate into the nucleus
- Important for transport of antigen into the nucleus

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13
Q

how can we prove that the signal for nuclear translocation allows active transport?

A

Example: T-antigen of the SV40 virus
- If the sequence is intact, the T-antigen is localised in the nucleus
- If the sequence is disrupted (mutation from lysine to threonine), there is no staining in the nucleus, so the T-antigen immunofluorescence remains within the cytosol

example 2: can put the required Lys, Arg, Pro sequence on a protein that doesn’t normally enter the nucleus, and then show that this protein now can be translocated

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14
Q

how can we prove that the nuclear translocation uses active transport?

A

Evidence:
- in cells, mRNA transport out of the nucleus is inhibited when cell is cooled to 4C
- ATP hydrolysis is required for active transport

In vitro import of protein into the nucleus:
- In the absence of ATP, the protein binds to the pore but the complex remains outside the nucleus
- add ATP and the proteins start to appear inside the nucleus

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15
Q

How are membrane proteins made?

A
  • 30% of human genome encodes membrane proteins
  • Proteins that make up plasma membrane, Golgi, ER, endocytic network are synthesised in the ER
  • All of the proteins that are secreted from cells such as growth factors are translocated into the ER for secretion
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16
Q

what characterises the rough ER?

A

Rough ER is characterised by ribosomes which are tightly associated with the ER membrane
- Close proximity of ribosomes to ER membrane

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17
Q

what are the 2 main ways in which newly synthesised proteins can be translocated into the ER?

A
  1. co-translational translocation
  2. post-translational translocation
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18
Q

what is co-translational translocation?

A

Co-translational translocation: protein enters lumen of ER as it is being translated in the ribosome
- Most common mechanism
- Coupling of translation to translocation
- This is why ribosomes need to be in such proximity with rough ER membrane

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19
Q

what is post-translational translocation?

A

Post-translational translocation: protein is fully synthesised in cytoplasm, and then is translocated into ER lumen (rarer)

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20
Q

what is the signal hypothesis of ER translocation?

A

In order for a translated protein to be recognised by translocation machinery, it requires a signal

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21
Q

what is the process of protein co-translational translocation into the ER lumen?

A
  1. When a ribosome is translating mRNA, if the mRNA is due to be translocated it will have a signal
  2. The translocator (sec61) will be tightly closed if it isn’t associated with a ribosome
    - ER has a different ionic composition to cytosol, meaning it is tightly controlled to ensure no leakage
  3. When signal of protein that needs to be translocated is detected, the ribosome interacts with the translocator, the signal is recognised, and the newly translated protein is slowly fed in through the pore
  4. The signal is then removed by a signal peptidase to cleave it off the translocator
    - Signal is then degraded
  5. The fully translated protein now exists in the lumen of the ER as a linear polypeptide, so has to fold using chaperones
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22
Q

how does translocation into the ER compare to nuclear pore recognition?

A

The recognition of the signal is by the receptor of the translocator which recognises a variety of signal sequences
- Less selective compared to nuclear pore recognition

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23
Q

how are membrane proteins inserted into the ER membrane via co-translational translocation?

A

example: single-spanning membrane protein with 1 TM domain
1. Start-transfer sequence starts the translocation by being recognised by sec61 translocator

  1. Newly translated protein is fed though the translocator
  2. Instead of going all the way through, it encounters a stop-transfer sequence, which is a hydrophobic part of the protein
    - The hydrophobic part allows the protein to remain anchored within the ER membrane
  3. The C-terminus of the protein is exposed to the cytoplasm, while the N-terminus remains in ER lumen

When transported to the plasma membrane, the protein will then have type I topology (N facing extracellularly, C facing cytoplasm)

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24
Q

what is type 1 topology?

A

when the N terminus of a transmembrane protein faces extracellularly, and the C-terminus faces the cytoplasm

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25
Q

what is type 2 topology?

A

when the N-terminus of a transmembrane protein faces the cytoplasm, and the C-terminus faces extracellularly

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26
Q

what is important about membrane protein topology?

A

Membrane proteins always have the same topology
- If the N-terminus is to outside of the cell, then this is always the case for that particular protein

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27
Q

how is a single transmembrane with a type 2 topology inserted into the plasma membrane?

A
  1. Signal sequence recognised by translocator
  2. C-terminus is fed through into the ER
  3. N-terminus remains in cytosol
  4. When the protein is transported to the plasma membrane, the C-terminus will face extracellularly, and the N-terminus will remain in cytoplasm, and so have a type II topology
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28
Q

what factors ensure proper protein folding in the ER?

A
  1. BIP chaperone associates with newly synthesised proteins to ensure proper folding
  2. quality control (QC) mechanisms within the ER ensure correct folding
    - post-translational glycosylation occurs in the ER to ensure QC
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29
Q

What is an example of BIP chaperone function in proper protein folding?

A

In antibody formation, the BiP remians associated until both light chains have been assembled correctly
- When BiP dissociates, the antibody can then be packaged into the vesicle

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30
Q

what happens if proteins are not folding properly in the ER?

A

If proteins are not folding properly, they are reverse-translocated into the cytoplasm and broken down

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31
Q

what are the consequenses of protein misfolding?

A

Defects in protein folding give rise to disease e.g. CFTR delta-F508
- If cells are making lots of proteins, QC machinery gets overwhelmed and proteins remain in the ER
- Excess of misfolded proteins stimulate the global unfolded protein response (UPR) which may lead to apoptosis

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32
Q

what is the unfolded protein response (UPR)?

A

UPR is a transcriptional programme where cells stop the translation of proteins and upregulate synthesis of chaperones by transcription factors
- The chaperones will then fix the protein folding
- If there are too many unfolded proteins, then apoptosis is activated
- This process must be balanced: can be useful against cancers but can also be harnessed by cancers

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33
Q

how do proteins enter the mitochondria?

A

Post-translational translocation
- the proteins are fully synthesised and then are translocated into the mitochondria using signal sequences
- Uses translocation proteins embedded in the outer and inner mito membrane

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34
Q

what key complexes are involved in protein translocation into the mitochondria?

A
  • TOM: Translocator of the Outer Membrane
  • SAM: Sorting and Assembly Machinery
  • TIM: Translocator of the Inner Membrane
  • OXA: Cytochrome Oxidase activity
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35
Q

how do proteins enter the mitochondrial matrix by post-translational translocation?

A
  1. N-terminal signal sequence is recognised by the TOM complex
  2. The protein translocates through TOM and TIM23
  3. Protein translocates through TIM23 into matrix
  4. Signal is cleaved off
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36
Q

what is the structure of the translocation signal into the mitochondrial matrix? how does it trigger translocation?

A

amphipathic alpha helix:
- one side of the helix is hydrophobic, the other is hydrophilic
- TOM receptors on the mitochondrial outer membrane recognises the polar structure rather than the amino acids

hydrophilic residues of the helix bind to the hydrophobic groove of the TOM complex receptor, leading to transloaction

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37
Q

How are mitochondria and bacteria linked?

A

Mitochondria are thought to have bacterial origin due to translocation mechanisms being similar
- Both mitochondria and bacteria have porins (made up of barrels of beta-sheets) in outer membrane for free exchange of metabolites and ions

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38
Q

how are proteins translocated into the bacterial membrane?

A
  1. TOM complex translocates the porin polypeptide chain
  2. chaperones help the polypeptide to assemble
  3. SAM complex helps it assemble in the outer membrane

In gram-negative bacteria, to insert porins in outer membrane, polypeptide is translocated into periplasmic space, periplasmic chaperones fold the polypeptide and then insert it into the outer membrane

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39
Q

how are proteins translocated into chloroplasts?

A
  • Translocation of proteins in chloroplasts also occurs post-translationally
  • Uses membrane potential and ATP to drive the process
  • Plant cells have both chloroplasts and mitochondria, so proteins are sorted accordingly based on selective signal sequence recognition
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40
Q

what mediates transport of protein through the secretory pathway between organelles?

A

vesicles and tubules
- organelles of the cell have interconnections for trafficking between them

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41
Q

what is anterograde and retrograde transport?

A

anterograde: forward trafficking from ER to Golgi
retrograde: backward trafficking from Golgi to ER

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42
Q

how are newly synthesised proteins moved via the secretory pathway?

A
  1. Protein is modified in ER
  2. protein is then packaged and sorted at the exit site into vesicles, as long as quality control system has checked they have been correctly folded
  3. Vesicles then enter Golgi – concentrated amounts of proteins
  4. Microtubules mediate movement of vesicles from ER to Golgi
  5. Vesicles bud from trans-Golgi network and fuse with plasma membrane
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43
Q

how are vesicles targeted to their destination?

A
  • When vesicles are targeted to their destination, their cargo will bud off the donor compartment (ER) and fuse with the target/acceptor compartment (Golgi)
  • The asymmetry of the membrane is maintained
  • Fusion is non-leaky

SNAREs - ensure right vesicle go to right place

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44
Q

what is the role of SNAREs in vesicle fusion?

A

SNAREs act as an address label on vesicles, ensuring that the right vesicle goes to the right place in the cell and fuses with the correct target membrane

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45
Q

how are the different transport vesicles coated?

A
  • Clathrin has a distinct hexagonal structure
  • COPII coat is formed by peripheral membrane proteins that are recruited from the cytoplasm onto the membrane - Forms specific structure which is important for vesicle formation
  • COPI coat contains alpha beta and epsilon subunits
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46
Q

what are the 3 essential components for all transport vesicle formation?

A
  1. GTPase
  2. Adaptor proteins - these recognise the cargo
  3. Coat - Clathrin, COPII, COPI
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47
Q

what are small GTPases?

A
  • GDP form usually in cytosol and is inactive
  • GTP form is membrane-associated and is active - when Ras is in GTP form, it activates downstream effectors
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48
Q

how are small GTPases regulated?

A

GEFs (guanine nucleotide exchange factors) convert GDP to GTP -> activates the GTPase

GAPs (GTPase activating proteins) hydrolyse GTP to GDP -> inactivates the GTPase

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49
Q

what is the founding member of the small GTPases?

A

Ras
-Ras is mutated in many forms of cancer – constitutively active Ras causes cancer

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50
Q

what are the propertoes of small GTPases?

A
  • Rabs, Arfs, Ran, Rho – characterised as molecular switches (on/off)
  • All between 20-30kDa
  • They have a GTPase domain and a domain which confers specificity
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51
Q

which small GTPase is essential for nuclear transport?

A

Ran

52
Q

how is Ran GTPase involved in nuclear transport?

A

Ran is found in the cytosol in its GDP inactive form:
1. When cargo needs to enter the nucleus, the cargo binds to nuclear receptors, and the cargo enters the nucleus

  1. There is lots of GEF localised to chromatin, meaning there is lots of active Ran-GTP in the nucleus
    - When cargo enters nucleus, Ran-GTP binds to the cargo and receptor
  2. Cargo then dissociates from the Ran-receptor complex and stays in the nucleus
  3. Ran-GTP bound to the receptor exits the nucleus, encounters GAP which hydrolyses the GTP, forming inactive Ran-GDP so the cycle can restart
53
Q

how is the unidirectionality of nuclear transport achieved?

A

The segregation of GEF in the nucleus ensures the direction of the nuclear transport:
- GDP form is in cytoplasm – allows cargo in cytosol to bind to nuclear receptors and enter nucleus
- GTP form is in nucleus – required to dissociate cargo from nuclear receptors and maintain cargo inside the nucleus

54
Q

where do COPII vesicles form?

A

COPII vesicles form at the exit sites of the ER.
- When newly synthesised are released to exit via the secretory pathway, they are packaged into COPII-coated vesicles

55
Q

what are the 3 key components of the COPII vesicles?

A
  1. GTPase: Sar1 (member of the Arf family)
  2. Adaptor: sec23/24
  3. Coat: sec13/31
56
Q

how is COPII vesicle formation activated?

A

When cargo needs to be sorted into coated vesicle, there is a cargo receptor on the ER membrane which is recognised by the adaptor protein:
- This only happens when Sar1 GTPase is activated

  1. Sar1 gets activated by GEF in the ER membrane, converting it from GDP to GTP-active form
  2. Sec23 binds to Sar1, Sec24 binds to the cargo - this allows recruitment of the adaptor complex
  3. Sec24 recognises a signal by the cargo receptor, allowing it to be incorporated into the COPII vesicle
    - Adaptor complex has a bowtie shape which allows it to recognise a curved membrane
  4. The exit signal on the cargo recognises the bud receptor, allowing the cargo to bind to the adaptor sec23/24 complex
    * Adaptor complex allows the recruitment of the COPII coat
57
Q

why is membrane recruitment crucial?

A

Membrane recruitment is crucial as adaptors must recognise the cargo the GTPase ensures the recruitment is to the correct membrane in the cell

58
Q

how are resident ER proteins excluded from the COPII bud?

A

Must package essential proteins that must enter secretory pathway while reducing the no. of resident proteins that may move through the pathway
- A high SA:V ratio means that less soluble ER resident proteins get trapped in the bud, and only the cargo can enter the bud

59
Q

what is COPII vesicle formation coupled to?

A

COPII vesicle formation is coupled to the quality control mechanisms in the lumen of the ER:
- Coat stabilises the bud
- When resident proteins are no longer associated with chaperones and properly folded, they can enter exit sites and be packaged

60
Q

how does COPII vesicle budding occur?

A
  • Sec23/24 recruits the COP2 coat to form the vesicle during budding
  • When the bud is fully formed, it pinches off to form a coated vesicle
  • Inside the vesicle, it is crowded with receptors and cargo to help exclude resident ER proteins and ensures that few of them escape through secretory pathway
  • If some resident ER proteins do escape, there are mechanisms to retrieve them
61
Q

what do the COPII coat proteins do for the vesicle?

A

they provide a structural scaffold to make the vesicle rigid and stable

62
Q

what must occur in the COPII vesicle to allow fusion to take place?

A

After budding, the coat must be removed in order for fusion to occur
- In sec mutants, there is an accumulation of coated vesicles under EM
- If the uncoating step is inhibited, the vesicles cannot fuse with target membrane and secretion is therefore blocked

63
Q

how can COPII vesicles be studied?

A

ER can be isolated from cells and used for reconstitution:
- Can reconstitute formation of COPII vesicles in-vitro
- ER is a large membrane component of cells
- When cells are broken open by homogenisation, ER membranes can be isolated
- Can run ER lysate membranes on a sucrose gradient
- Rough ER will centrifuge to equilibrium and can be sucked out with a pipette as microsomes

64
Q

how can reconstruction experiments allow us to understand what makes a COPII vesicle?

A
  • They took an ER membrane preparation and identified ribophorin, a known ER resident protein, in green
  • COPII vesicles were labelled in red as it contained a known COPII cargo called p58
  • known that COPII vesicles would have a different protein and lipid composition to the ER membrane

assay: if ER membranes are run on a sucrose gradient via centrifugation, they will pellet at a particular sucrose concentration, while the COPII vesicles will pellet at a different concentration
- can clearly separate ER and COPII vesicles
- cytosol, ATP and GTP needed to be added, a band of p58 is seen, showing COPII is formed by these molecules
- ribophorin was added to show that COPII vesicles are free of ribophorin from the ER

65
Q

what are the key COPII components?

A
  • GTPase is Sar1, GEF is Sec12
  • Sec23/24 - adaptor
  • Sec13/31 – coat
  • ATP and GTP
66
Q

how are GTPases involved in coat assembly/disassembly?

A
  1. Inactive, soluble Sar1-GDP can be recruited by GEF to be converted to active Sar1-GTP
    - Sar1-GTP can recruit Sec23/24 adaptor from the cytosol to recognise the cargo, and sec13/31 coat
  2. Sec23 adaptor can act as a GAP for active-Sar1:
    - GAP activity is enhanced following recruitment of the coat Sec13/31
    - Coupling of Sec13/31 coat formation and budding from ER, with the inactivation of Sar1-GTPase, leading to coat disassembly as the Sar1 is hydrolysed from GTP to inactive GDP
    - Active Sar1-GTP is needed initially to form the coat and recruit the adaptor
  3. Once the vesicle has budded, sec13/31 coat makes the sec23/24 adaptor protein a better GAP, which hydrolyses the Sar1-GTP to Sar1-GDP, leading to coat disassembly
67
Q

what are the consequences of mutant, constitutively active GTPases?

A

Sar1-GDP mutant: sequesters GEF so cannot exchange GDP to GTP
- Off all the time
- Expression of Sar1-GDP mutant inhibits COPII formation

Sar1-GTP mutant: sequesters GTPase cannot hydrolyse GTP to GDP
- On all the time
- Mutant GTPases often have dominant-negative effects

Cycles of GTPase activity are important

68
Q

what are the GTPase, coat and cargo of COPII vesicles?

A

key components:
- GTPase = Sar1
- coat = COPII
- cargo = newly synthesised proteins

COPII packages newly synthesised proteins which bud from the ER and move through the secretory pathway

69
Q

what are the GTPase, coat and cargo of COPI vesicles?

A

key components:
- GTPase = Arf1
- coat = COPI
- cargo = retrieved proteins and newly synthesised proteins

COPI vesicles move material both anterograde and retrograde
- retrieves escaped proteins and carries them backward (retrograde)
- moves newly synthesised proteins forward (anterograde)

70
Q

what are the GTPase, coat and cargo of TGN clathrin-coated vesicles?

A

GTPase = Arf1
Coat = clathrin
Cargo = lysosomal proteins and regulated secretory proteins

if lysosomal proteins are targeted to the endosomal pathway, these proteins are packaged into clathrin-coated vesicles

proteins which undergo regulated secretion are also packaged into clathrin-coated vesicles

71
Q

what occurs at the TGN?

A

at the TGN, material is sorted either directly to plasma membrane (constitutive secretion) or targeted to the endosomal pathway (e.g. lysosomal protiens)

72
Q

what are the GTPase, coat and cargo of plasma membrane clathrin-coated vesicles?

A

GTPase = unknown
coat = clathrin
cargo = endocytosed material

at the cell surface, clathrin-coated vesicles of the plasma membrane take up endocytosed material

73
Q

how can cargo in vesicles be detected and purified?

A

Cargo in vesicles can be detected by EM, and purification of the vesicle using mass spectrometry

74
Q

what are adaptor proteins?

A

These recognise cargo signals and select cargo to ensure specificity
- they concentrate the cargo into budding vesicle, and exclude material that does not express this signal
- they link the vesicle coat to the ER membrane – coat cannot directly bind to membrane, so adaptor acts as a link
- adaptors recognise motifs in the cytoplasmic domains of the membrane proteins

75
Q

how are adaptor proteins organised in the cell?

A

adaptor complexes show a precise subcellular localisation which can be seen with immunofluorescence
-AP1 found on endosomes and TGN - forms transport vesicles
- AP2 found at plasma membrane
- AP3 found at TGN and lysosomal-related organelles

76
Q

what allows adaptor proteins to go to the right location?

A

targeting motifs ensure the adaptor complexes go to the right place
- the adaptors recognise motifs in the cytoplasmic domains of membrane proteins

77
Q

why are cytoplasmic domains important for adaptors?

A

Cytoplasmic domains contain different motifs/signals to allow specific interactions with adaptors
- these can act as sorting signals in endocytic proteins
- e.g. tyrosine-based motifs, ubiquitin

78
Q

how does AP2 recognise peptide motifs?

A

AP2 has mu and delta subunits which can recognise sorting signals to bind to transferrin receptors:
- the 2 large subunits have flexible hinge-appendage domains which can interact with other proteins
- the beta-2 subunit of AP2 binds to clathrin
- mu-2 and sigma-2 subunits recognise signals in transmembrane proteins, ensuring specificity of different adaptor proteins

79
Q

what is AP2?

A

it is a major Clathrin adaptor protein involved in endocytosis

80
Q

what are the key points on adaptor proteins (summary)?

A
  • Recognise and select cargo to ensure specificity
  • Link vesicle coat to the membrane
  • Recognise motifs in the cytoplasmic domains of membrane proteins
  • Targeting motifs allow the adaptor complexes to go to the correct location
  • Endocytosis: AP2 is a major Clathrin adaptor
  • Other adaptors of Clathrin allow cells to select what they internalise
81
Q

when there are defects in membrane trafficking, why are only some tissues affected

A

There is redundancy of proteins expressed, and different versions of the same protein, so that if one tissue is affected, not all others are – do not have one copy of each protein
- Tissue specificity of protein expression

82
Q

what is cranio-lenticulo-sutural dysplasia?

A
  • issue with ossification of bone and hypermobility issues
  • caused by a mutation in sec23 -> abnormal ER to Golgi trafficking
  • problem with depositing collagen to the ECM
83
Q

what mutation in sec23 causes CLSD?

A

single amino acid change from phenylalanine to leucine in sec23
- mutation has such a strong effect because it is involved in the secreotyr pathway

84
Q

what did immunofluorescence studies on CLSD-patient fibroblasts show about sec23?

A

wildtype fibroblast showed sec23/24 exit sites forming and showed patches of fluorescence associated with ER

mutant CLSD fibroblast showed distended ER and larger fluorescent patches, with sec23 surrounding those patches
- suggested problem with COPII vesicle packaging

85
Q

what did observations of the CLSD-patient fibroblasts under EM show abiout the sec23-mutant COPII vesicles?

A
  • Wildtype fibroblasts showed classic ER tubules
  • Homozygous patient fibroblasts have huge ER membrane distension
  • Heterozygotes have some distension, but not as bad as homozygous
  • Suggested an issue with ER function and COPII vesicle formation
86
Q

what is the process of a reconstiution assay in understanding what makes a COPII vesicle?

A
  • ER starting material is measured by the presence of the resident protein ribophorin
  • ER is then incubated in conditions that COPII can form (cytosol, ATP, GTP)
  • Formation of COPII is measured by p58
  • Run on sucrose gel, isolate the fractions and observe p58 vesicles and ribophorin ER
  • Allowed them to dissect what is needed in the cytosol to make COPII
87
Q

what did the liposome binding assay determine about the sec23 mutant involved in CLSD?

A
  • binding of mutant sec23a to liposomes was unaffected
  • western blots showed that the sec23/24 mutant and wildtype bound equally well to GTP-Sar1

showed that the mutant sec23 did not affect the initial recruitment of sar1

88
Q

how was the sec23 mutant determined to have an effect on COPII formation?

A
  • when ER is incubated alone with no additional components, no COPII vesicles are formed
  • if ER is incubated with lots of cytosol, there is no COPII formed as there is no energy available
  • if ER is incubated with high cons of cytosol, ATP and GTP, cargos appear in the COPII vesicles

cytosol can be made limiting if we add lower amounts of available adaptors or coats
- no COPII vesicles form under these conditions
- if some coat is added, some COPII form
- most COPII is formed under limiting cytosol with the wildtype sec23/24, wildtype adaptor and wildtype coat

if everything wild type is added back, but the sec23 mutation remains, there is a clear reduction in number of COPII vesicles formed

89
Q

how was zebrafish used to study the sec23 mutation in CLSD?

A

Zebrafish used as an in vivo model:
- used morpholinos to knockdown sec23a in the zebrafish
- they looked for the staining of sec23a, and it was found particularly in neurocranial tissues (tissues which were affected in the patients)
- in the sec23-knockout zebrafish, the neurocranial structures are less defined (P) compared to the wildtype (Q)

provided supporting evidence that the mutation gives rise to these musculoskeletal and ECM collagen deposition problems – defect in movement of cargo via secretory pathway

90
Q

how does packaging of large cargo such as collagen occur?

A

collagen is synthesised as procollagen, which are very large filaments and would be unable to fit into COPII
- modifications of the COPII coat help to sort procollagen into tubular structures for transport

91
Q

how does the sec23 mutation lead to CLSD?

A
  • sec23 mutation causes defects in COPII machinery, leading to defects in large cargo deposition
  • so if COPII is unable to form due to the sec23 mutation, then collagen cannot be sorted into tubular structures and then deposited
92
Q

what is Rab?

A

rab is a member of the Ras-GTPase family
- it has distinct subcellular localisation
- it cycles between active-GTP in the membrane and inactive-GDP in the cytosol
- GEFs and GAPs are used to cycle rab activity
- 60 subtypes of Rab, most are ubiquitously expressed and used in most tissues
- they are all associated with specific membrane types e.g. rab1 is associated with ER membrane

93
Q

what is the main function of Rab?

A
  • it is required for fusion and other trafficking functions
  • regulates specific trafficking steps
94
Q

what is the role of Rab in fusion?

A
  • Rab-GTP helps to recruit a tethering protein
  • The tethering protein has a coiled-coil domain
  • The tether facilitates the formation of the SNARE complex by pulling the vesicle close to the membrane
95
Q

what is the process of the rab cascade in movement of cargo between organelles?

A
  • Rab5 regulates the early endocytic pathway by delivering content to late endosomes
  • Late endosomes are associated with rab7
  • Movement of cargo targeted for destruction in liposome moves from rab5 compartment to rab7 compartment via a cascade process

Rab5 is activated by GEF to form with GTP, and when cargo moves to late endosome, the GAP for rab5 recruits the GEF for rab7 to pass the cargo onto the next section

96
Q

what are examples of Rabs in disease?

A

Example 1:Charcot-Marie-Tooth 2B (neurodegenerative disease)
- Rab7a missense mutations lead to excessive activation, reduced autophagic flux, premature neurotrophin receptor degradation, inhibition of neurite outgrowth
- Rab7 is a late endosomal rab
- The mutation causes it to be constitutively active

Example 2: Harnessing of rabGEFs by pathogens
- Legionella pneumophila can recruit rab1 to the cell surface to create an ER-like compartment where it can replicate

97
Q

what is the role of smooth ER?

A

it is the site of lipid synthesis and important for calcium storage
- it maintains the lipid composition of intracellular fluid compartments e.g. cholesterol needed at PM
- it moves calcium throughout the cell e.g. for muscle contraction

98
Q

what does smooth ER require to function?

A

Intermembrane contact sites (MCS)

99
Q

how were MCS identified?

A

First identified in 1957 by Porter and Palade studying carcoplasmic reticulum in muscle
- They studied ER of muscle cells via microscopy
- EM showed cell contact sites that are electron dense
- Electron-dense areas represent that lots of protein is localised in that area
- Close connection between SR and plasma membrane

100
Q

how does ER use MCS?

A

The ER makes contact with many different organelles in the cell
- smooth ER has contact sites with many intracellular organelles for transport of lipids and calcium
- ER spreads throughout the cell to organelles and the plasma membrane
- ER-plasma membrane (PM) contact sites, ER-mitochondria, ER-Golgi etc

101
Q

are MCS caused by membrane fusion?

A

There is no evidence that these connections are due to membrane insertion/fusion:
- close connection/proximity, but no fusion

102
Q

why does smooth ER not have ribosomes?

A
  • wherever the ER contacts an organelle, there is an exclusion of ribosomes
  • this allows the smooth ER to come in close proximity with organelle membranes
103
Q

how can smooth ER structure be studied?

A

EM shows exclusion of ribosomes

104
Q

how can smooth ER interaction with organelles be studied?

A

live cell imaging can show dynamic interactions of smooth ER
e.g. role of ER in mitochondrial fission

105
Q

what is the role of smooth ER in mitochondrial fission?

A
  • During mitochondrial fission, the ER tubule gradually winds around the mitochondrion, forming a loop
  • The ER contributes to the breaking apart of the mitochondria during fission
  • MCS contribute to the fission/fusion process of mitochondria
106
Q

What is CLEM and what can it be used tfor to study?

A

Correlative light and electron microscopy:
- CLEM allows localisation of fluorescently labelled proteins to MCS
- Combines the power of light microscopy to observe immunofluorescence with the resolution power of EM
- Can capture the dynamics and stabilise it for visualisation
- Look at areas of high fluorescence under LM and mark with dot
- Use EM to visualise the area marked by dot in higher detail to see cell ultrastructure

107
Q

what is the structure of an ER MCS?

A
  • Ribosomes are excluded from the contact sites
  • The membranes are very close together (10-80nm)- Distance varies due to the nature of the proteins that link the 2 membranes
  • ER contacts can be short- or long-lived
  • Muscular SR contact lasts for the lifetime of the cell – muscle needs to continuously use the MCS
108
Q

how do proteins link the 2 membranes of an MCS?

A

MCS are connected by long multidomain proteins which stretch out and hold the membranes together
- The domains recognise different lipids on the membrane to hold the membranes together

109
Q

what tethers can hold MCS together?

A
  • Can be protein-protein
  • Can be protein-lipid via lipid-binding domain
  • Distance usually 30nm - No less than 10nm distance between the membranes, so no fusion
  • Inhibits fusion – membranes need to be 1-2nm from each other + lipid bilayer disruption
110
Q

what is the main key point about MCS formation?

A

fusion is inhibited as the membranes are no less than 10nm apart
- membranes need to be 1-2nm from each other, plus lipid bilayer disruption, in order for fusion to occur
- MCS does not involve lipid bilayer disruption, and membranes are kept >10nm apart

111
Q

what is the key criteria of MCS?

A
  • Membranes aren’t close enough to fuse
  • Water is not excluded, so fusion is inhibited
  • Must fulfil and function
  • They must have a microdomain with a defined protein and lipid proteome
  • They are raft-like, enriched in sterols (lipids) to aid rigidity
112
Q

what is Osbp?

A

Osbp is both a tether and a lipid transfer protein: must fulfil a function
- Transfers cholesterol into the ER
- involved in MCS formation

113
Q

what are the main fucntions perfomed at MCS?

A

To provide platforms for:
* Calcium mobilisation
* Lipid transfer
* Signalling
* Organelle division

114
Q

how is calcium conc regulated in the cell for muscle function?

A

Muscle requires calcium mobilisation as well as for other functions:
- Intracellular calcium is kept low(1nM)
- Intra-organelle and intracellular calcium varies
- Newly formed endosomes are 1nM – they are pinched off from cell surface so similar to extracellular conc
- Early endosome is 0.5uM
- High concs of calcium in ER at 500uM
- Lots of calcium in ER lumen, and much less in cytosol and other organelles

115
Q

what is the sarcoplasmic reticulum?

A

Specialised ER for handling calcium transients required for muscle contraction

116
Q

what is the role of the SR in calcium mobilisation for muscle contraction?

A
  • When an AP arrives at a muscle cell, there is mobilisation of calcium from the SR
  • This is key for muscle contraction
  • This occurs via the close connection between the T-tubules of the plasma membrane and the calcium stores within the SR
  • This is long-lived ER contact site
117
Q

how do the SR MCS diffr in skeletal and cardiac muscle?

A

T-tubules from PM and SR form a triad (skeletal) and dyad (cardiac) in a very close connection

118
Q

how is calcium mobilised and replenished into SR stores?

A

Stim1 is a calcium sensor in the SR:
1. Under resting conditions with lots of calcium in SR, Stim1 is monomeric

  1. When calcium levels fall in the SR, there is an oligomerisation of Stim1 - The protein comes together to an oligomer which travels to the tip of an SR tubule
  2. At the tip, Stim1 interacts with an Orai1 channel on the plasma membrane triad
    - Orai1 channel is enriched in a particular lipid called PIP2
    - Phosphoinositides are important lipids in cell signalling and in conferring organelle identify
    - PIP2 defines the identity of the MCS
  3. PIP2-enriched Orai1 channel makes it recognisable with Stim1, forming an MCS
  4. Calcium can now be transported into the ER to replenish the stores
119
Q

what is the process of cellular uptake of cholesterol?

A
  • Low density lipoprotein (LDL) is taken up into the cytosol by LDL receptors as cholesterol
  • The cholesterol is delivered through the endocytic pathway via vesicular trafficking to the early endosome via fusion
  • The cholesterol enters late endosome, into lysosome, where it is hydrolysed and released as free cholesterol
120
Q

how are MCS important in movement of free cholesterol into the ER?

A

ER: site of membrane lipid synthesis – cholesterol needs to be delivered to the ER to be sorted to the plasma membrane
- Contact sites allow the non-vesicular transfer of lipid
- Lipid transfer is unidirectional – e.g. from lysosome to ER
- Lipid transfer proteins (LTPs) have hydrophobic grooves to protect the lipid from the soluble environment of the cytosol
- LTPs use lipid or ion concentration gradients to promote lipid transfer into the ER
- They use a gradient of ions in one direction to promote the transfer of lipids against its conc gradient into the ER (antiport)(counter-transport)

121
Q

what happens to cells if someone has a diet deficient in cholesterol?

A

if there is a diet which is deficient in cholesterol, genes for cholesterol synthesis get switched on, so that the cholesterol sensors in the ER activate cholesterol synthesis

122
Q

what may happen if there are defects in MCS tethering proteins?

A

can lead to disease e.g. Niemann Pick disease C

123
Q

what is Niemann Pick Disease C?

A

NPDC is the accumulation of lipids in the spleen, liver, lungs, bone marrow and brain
- It is an inherited homozygous condition
- Sphingomyelin accumulates in lysosomes
- Mutation in a transmembrane protein which is important for MCS involved in movement of cholesterol from lysosome into ER
- Leads to red globules of fat in lysosomes

124
Q

how are MCS involved in cell signalling?

A

MCS help regulate transport of the epidermal growth factor receptor (EGFR) into multivesicular bodies (MVB)
- EGFR is a receptor tyrosine kinase (RTK)
- When RTKs are activated by ligands, their cytoplasmic domain becomes phosphorylated, and they initiate a signalling cascade
- To stop this signalling, RTKs need to be dephosphorylated and downregulated in the lysosome
- EGFR is dephosphorylated by PTP1b (protein tyrosine kinase 1B)
- The tyrosine kinase dephosphorylates the EGFR at the endosome, leading to the EGFR signalling to be switched off, and the receptor is incorporated into MVB for degradation
- Key in regulation of EGFR

125
Q

how are MCS involved in organelle fission?

A
  • Mitochondria fission site is marked by a fission enzyme
  • The ER membrane surrounds the fission enzyme, triggering the fission event
126
Q

what diseases are linked to MCS

A

TDP-43 protein is pathologically linked to ALS:
* It regulates ER-mitochondria contacts
* It is mutated in ALS

Disease-associated mutations in hereditary spastic paraplegia, linked to REEP1 protein
* Mutation of REEP1 leads to disruption of ER-mitochondria contacts

Presenilins involved in calcium exchange between ER and mitochondria
* When these are mutated, there is a disruption in calcium exchange from ER to mitochondria

Molecular cellular biology (13 decks)

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