liz Smythe - vesicles and stuff

Question 1

anterograde vs retrograde?

Answer 1

Anterograde movement - moving forward in the secretory pathway

Retrograde - moving backwards to the organelle from whence you came!

Question 2

basics about vesicles?

Answer 2

vesicles bud off donor, fuse with membrane of target

Asymmetry of membrane is maintained and fusion is not leaky

Snares are the address label, all vesicle require snares

Vesicles have a distinct coat

Question 3

what are the three essential components for vesicles and their roles?

Answer 3

Small MW GTPase
Ras is the most well known, but there are also Rabs, Arfs, Ran etc…
Characterised as ‘switches’, on/active with GTP vs off/inactive with a GDP

Go from inactive to active via GEFs (guanine exchange factors)
Active to inactive via GTPase activating protein, a GAP, as their intrinsic GTPase activity is low and needs a boost

Adaptor proteins
Recognise and select cargo (ensures specificity)
Links the coat to the vesicle membrane

Coat proteins
Stabilise the vesicle as it buds, acting as a kind of structural scaffold

Question 4

what are the three components of the COPII vesicles?

Answer 4

GTPase = ‘Sar1’ of the Arf family

Adaptor = Sec23/24 (sec tells us it was originally found in yeast and is conserved between yeast and many species including humans)

Coat = Sec13/31

Question 5

explain how COPII vesicles form

Answer 5

ER Exit Site (ERES):
COPII vesicle formation begins at specific regions of the ER called ER exit sites (ERES)

Initiation of COPII Vesicle Assembly:
The first step involves the activation of small GTPase protein, Sar1, by the addition of GTP (guanosine triphosphate)

Sar1 is initially associated with the ER membrane in an inactive form (Sar1-GDP). The GEF Sec 12 (also associated with ER membrane) is needed to activate Sar 1

Recruitment of Coat Proteins:
Activated Sar1 (Sar1-GTP) recruits the COPII coat protein complex to the ERES. the inner coat is made up of adaptor proteins - Sec23/24 and and the outer coat is made up of proteins Sec13/31
Note, it is Sec 23 that binds to the GTPase Sar-1

Cargo Selection and Binding:
Cargo receptors in the ER membrane recognise and bind to the soluble proteins in the ER to be transported - the ‘cargo’. These receptors have signals on the other end recognised by the adaptor proteins - specifically Sec 24
Mechanisms are in use to retrieve escaped resident proteins

Vesicle Budding:
The COPII protein coat provides stability - a ‘structural scaffold’ - as the membrane invaginates to form the vesicles

Question 6

how are ER resident proteins excluded?

Answer 6

Note - Want to exclude ER resident proteins from the being taken up in the vesicles, so for soluble proteins, a high Sa:V for the bud wont trap too many resident proteins (the cargo you want is bound to cargo receptors in the membrane - so more SA = more cargo you want)

Mechanisms are in use to retrieve escaped resident proteins

Question 7

how do COPII vesicles pinch off?

Answer 7

Sar1 hydrolyses GTP to GDP, leading to a conformational change that helps release the COPII coat from the vesicle.

It is actually Sec 23 - the adaptor protein, that functions as a GAP for Sar 1, assisting in the hydrolysis of GTP

The mature COPII vesicle, now devoid of its coat, is pinched off from the ER membrane and is ready for intracellular transport

Uncoating is necessary for SNAREs to do their thing

Question 8

aside from stabilising the vesicle as it forms, what do the coat proteins Sec 13/31 do?

Answer 8

The outer coat proteins - Sec 13/31 - actually enhance Sec 23’s GAP abilities

Question 9

how were the components of COPII vesicles discovered?

Answer 9

using reconstitution studies

To separate smooth and rough ER use a sucrose gradient - they’ll collect at different concentrations (though we call them microsomes at this point because ER membranes vesiculate)

Identified ER membrane via a known ER-membrane bound protein
identified/followed COPII vesicles via the cargo p58

Through some experiments they figured out what they needed to make vesicles - ATP, GTP and cytosol

What components of the cytosol are needed?
Essentially added the Sec proteins identified in yeast as necessary for vesicular transport in different combos to identify Sar 1 (and Sec 12) Sec 23/24 and Sec 13/31

Question 10

what are the two kinds of GTPase mutants?

Answer 10

GDP mutant - Permanently inactive - results in a sequestering of the GEFs as these like to bind to each other. This means GEFs aren’t available to activate any non-mutant versions
So overexpression of sar1-GDP inhibits COPII formation

GTP mutant - permanently active - cannot hydrolyse GTP - in case of COPII, Sar 1-GTP must be inactivated for uncoating - so you’d get an accumulation of COPII vesicles (at the ER?)

Question 11

why are GDP mutants useful?

Answer 11

Experimentally this can be used to figure out at what point/trafficking step is a particular GTPase needed

Question 12

importance of protein sorting and compartmentalisation?

Answer 12

Organelles all have unique protein compositions, so these need to be sorted when making organelles

Compartmentalisation allow cells to perform a much wider range of functions

Question 13

nuclear transport -
what are nuclear pores like?

Answer 13

Nuclear pores - made up of 30 diff. Proteins called nucleoporins. Each has a central plug and 8 subunits

Question 14

what do nuclear pores do?

include an example of what goes in and out

Answer 14

gate in and out of nucleus -
The pores let in 1000s of molecules/second, e.g. ribosomal proteins to combine with rRNA to make ribosomes

Question 15

what size molecules can pass through the nuclear pore?

Answer 15

Small molecules (MW<5000 Da) are freely diffusible

for molecules around 17,000 Da diffusion takes 2 min, 44,000 takes 30 min

At 60,000 diffusion isn’t possible, >60,000 Da proteins need ATP and a signal to enter

Question 16

what signal is required for nuclear transport?

Answer 16

Acidic linear signals, lots of lysines, Arg and Pro etc… act as this signal, recognition sites for nuclear transport

Question 17

how can you prove a certain sequence is the signal for a certain kind of transport?

Answer 17

something (e.g. T antigen of SV40 virus but specifics not needed) that normally enters nucleus - mutate the suspected signal sequence - should accumulate in the cytoplasm

OR take something not normally able to enter the nucleus, add the signal sequence, and see if now it ends up in the nucleus

Question 18

how can you prove transport is active?

Answer 18

mRNA can be inhibited from nuclear transport by cooling to 40C (so requires energy)

In vitro assays - in absence of ATP proteins normally transported into the nucleus are not. Adding ATP shows the proteins moving into the nucleus as expected

Question 19

what are the two methods of ER transocation?

Answer 19

co-translational translocation - occurs as the protein is being translated/made

post-translational translocation, once the protein is completed, e.g. inserting proteins into the ER membrane

Question 20

how does co-translational translocation work for the ER?

Answer 20

Signal sequence on N terminus of protein results in protein being threaded through SEC 61 - a protein that forms a pore on ER

SEC61 is very tight on the membrane and only opens in response to a signal sequence

Once protein is inside, signal peptidase snips off the signal sequence

SEC61 allows hydrophilic proteins to pass through the hydrophobic membrane

Question 21

how does post-translational translocation into the ER membrane work?

Answer 21

If a protein is meant to go into the membrane, the transmembrane domain sequence in the PP chain is seen as a stop sequence by SEC 61, meaning the trans-mem domain stops across the membrane.

Signal peptidase snips off the original N-terminus signal sequence (obvi not the TM domain)

Question 22

how do post-translational translocations type 1 and type 2 differ?

Answer 22

type 2 is when the signal sequence isn’t cleavable, and is somewhere withing the protein, not at the N-terminus like in type 1

Question 23

how does folding of a protein occur?

Answer 23

Occurs in ER lumen via chaperone proteins (assist in folding, things like disulfide isomerases as these bonds can’t form in the ‘reducing’ cytoplasm)
Key chaperone = BiP, most well known. Only dissociates from e.g. an antibody being made, when the antibody is complete (quality control)

Question 24

what happens if a cell gets an accumulation of unfolded proteins?

Answer 24

e.g. in cystic fibrosis when Cl channel is prevented from reaching the plasma membrane, you get UPR (unfolded protein response) = protein production is shut down (mostly) while more chaperones are made to deal with this

Question 25

how do you get proteins into the mitochondrial matrix?

Answer 25

(Like proteins and the ER) a mitochondrial protein once translated has a signal sequence on N terminus that allows it to dock to the mitochondrial membrane, via a receptor in the pore TOM complex (found in the outer membrane), the protein can then be fed in.

Now in the intermembrane space, it is fed through a TIM 23 complex into the matrix.
Signal sequence is cleaved via signal peptidases, you now have a mature protein in the matrix

Question 26

how do you get proteins into the mitochondrial outer membrane?

Answer 26

Feed protein through TOM complex, this time not going into the matrix, so here in the intermembrane space it’s coated in chaperone proteins, a SAM complex then inserts the protein into the outer membrane

Question 27

is getting proteins into chloroplast the same as mitochondria?

Answer 27

same principle, different molecular detail (i.e. different proteins used)

TOC instead of TOM, TIC instead of TIM

Question 28

give an example of structure, not sequence, being the signal for transport

Answer 28

Cytochrome C oxidase -
An example of a protein being translocated into the mitochondria

The signal here isn’t only an amino acid sequence, but its amphipathic helical structure, resulting in polar Aas facing away from the receptor, on the outside with hydrophobic residues facing the receptor. The STRUCTURE is what is taken as the signal to let the protein into the mitochondria

Question 29

what are the functions and abilities of adaptor proteins?

why are there so many?

Answer 29

Recognise and select cargo, ensuring specificity

Link the coat to the membrane of the vesicle

Recognise structural motifs/A.a sequences of membrane proteins, in order to fuse with the correct membrane

At plasma membrane, there are so many things around that may need to be taken in/out of the cell, so many different adaptor proteins are there in order to select what is needed

Question 30

what are Rabs? where are they found?

Answer 30

Member of the Ras superfamily. they are small GTPases (so go from GDP form as inactive to GTP form as active), and their role is to assist in vesicle trafficking in multiple ways, one of which is assisting in vesicle docking

Most Rabs are expressed in all cells (mostly), with very specific homes within the cell (distinct subcellular localisation, e.g. rab 1 at ER)

Question 30

AP2 is the adaptor protein for clathrin vesicles. what is it’s structure like?

Answer 30

It’s two small subunits (u2 and sigma2) recognise cargo
Also have a motif to recognise clathrin coat

Question 30

how do Rabs show that movement of cargo in a cell is highly regulated and specific?

Answer 30

because there are many Rabs, all with specific subcellular locations

Question 30

what is a Rab cascade/ what does it actually do?

Answer 30

a Rab cascade is when one of a Rab’s effectors that it activates, is another Rab (hence cascade)

an example of how it is useful is e.g. in the endocytic pathway, Rab 5 is responsible for the movement of cargo from the plasma membrane to the early endosome

one of Rab 5’s effectors is Rab 7, which is responsible for movement of cargo from the early endosome to the late endosome

Question 31

Cranio-lenticulo-sutural dysplasia
When the fontanelle - soft spot on front of skull - doesn’t close properly
Discovered to be a trafficking defect, a single mutation of Phe to Leu in Sec-23a (COPII adaptor protein) So thought to be a defect in ER to golgi trafficking (COPII)

1. what was seen when the ER were viewed?
   2.

Answer 31

looking at healthy patients -

ER were nice and reticular, for when an ER marker protein was stained, the SEC23A protein of interest was stained, and also when collagen(which should be transported from ER in COPII vesicles) was stained

mutant = congested and swollen for all three proteins, suggesting issues in COPII formation resulting in inability to traffic collagen

Question 31

the Rab cycle - how does it work? include the roles of the GDI protein

Answer 31

GTP forms are membrane associated, GDP forms are found in the cytoplasm

a GEF on ER membrane recruits a GDP rab from cytoplasm, GEF activates it to GTP form. this becomes part of a vesicle budding form the donor compartment

Rab effectors/tethering proteins assist in docking (by grabbing onto the Rab GTP associated with the vesicle), to help snares with fusion of vesicles to the target membrane. The Rab effectors increase efficiency

After fusion, GAP comes along and assists in hydrolysis of GTP to GDP to inactivate Rab

A chaperone called GDI then extracts GDP Rab from the target membrane. Importantly, it tucks away Rab’s fatty acyl tail (used to cycle on and off of membranes), to prevent it precipitating in the soluble environment of the cytoplasm

Question 32

if this mutation in SEC23 affected COPII vesicles, why were only certain tissues affected?

Answer 32

The reason why is ‘redundancy’ - there’s no problem with things like digestion, insulin secretion, other tissues etc… because other ‘paralogues’/isoforms are expressed at higher levels and compensate

Question 33

how is large cargo, like collagen fibres, packed?

Answer 33

won’t fit into regular COPII vesicles

Accessory proteins that enable COPII vesicles to rearrange themselves in order to fit larger cargo in like collagen fibrils.

Still up to debate, but it is theorised that ‘transient membrane tunnels’ form to allow the collagen to move to the next stage in the secretory pathway

Question 34

chloridaemia is a disease that causes issues with the retina resulting in blindness in men - what is it?

Answer 34

defect in a gene on X chromosome encoding Rab Escort Protein 1 (REP-1)

this normally ensures newly synthesised rabs interact with the machinery that puts the fatty acyl tail on

treatment = gene therapy using a virus to deliver the functional gene

Question 35

Griscelli syndrome results in hemophagocytic syndrome and albinism. what is it?

Answer 35

Rab27a mutation affects platelets and causes hemophagocytic syndrome
Delivery of material to secretory granules in immune cells uses similar machinery for secreting melanin in melanocytes, so albinism is also seen

Question 36

Charcot-Marie-Tooth neuropathy - trafficking for a receptor important in neuronal development is dsirupted. Why?

Answer 36

can be caused by several mutations, but is often due to defective Rab 7, responsible for late endosomal trafficking to lysosomes

Question 37

what are membrane contact sites? how do they appear?

Answer 37

The membrane of one organelle is in close contact with the membrane of another, but theres no membrane insertion or fusion

In EM, these sites appear as electron-dense black spots. Starvation in mice lead to disruption of these sites

Question 38

which organelle forms the most membrane contact sites?

Answer 38

idk if its the MOST, but the ER’s reticular structure spreads throughout the cell, its membranes making contact with most organelles throughout the cell, including association with plasma membrane

Question 39

what technique is useful when viewing membrane contact sites?

Answer 39

CLEM - colourative light and electron microscopy -

Allows you to first look at something fluorescent e.g. something you’re interested in, then mark and focus on the areas of your sample that fluoresced to view them in much higher resolution via electron microscopy. Live imaging is also a technique used a lot in this area

Question 40

how are membrane contact sites formed/what are they like? how long do they last?

Answer 40

ER - always a smooth area, so ribosomes are excluded
Like any membrane domain, they have defined lipid and protein compositions, often high in sterols

Many proteins anchored in the ER, extend and interact with components of the other organelle. NOT a covalent interaction as it can be transient (short-lived, or long-lived, the interaction is just broken down at different rates
The membranes are super close (10-80nm)

Question 41

the proteins that interact between different organelle membranes can have multiple functions - like what?

Answer 41

they act like like tethers (protein-protein interaction). These tethers can often also function to move molecules, e.g. transferring lipids…

note - Can have protein-lipid interactions or protein protein

Question 42

name four functions of membrane contact sites

Answer 42

Provide a space for calcium mobilisation, lipid transfer, signalling, organelle division

Question 43

how is calcium important in relation to the ER?

Answer 43

The ER is used as a calcium store, and Ca is then used as a second messenger, and in mechanisms like muscle contraction (AP from neuron results in release of calcium from sarcoplasmic reticulum, resulting in muscle contraction)

Question 44

how are membrane contact sites involved in replenishing the cells calcium stores?

Answer 44

STIM is located in the ER membrane and serves as a calcium sensor. When ER calcium levels decrease, STIM undergoes a oligomerisation and migrates to a particular area of the ER membrane *** involved in a contact site with the plasma membrane (which is defined by presence of PIP2 lipids, a kind of phosphoinositides which are very rare)

STIM interacts with ORAI proteins at the plasma membrane, leading to the opening of the ORAI calcium channels. This allows extracellular calcium to enter the cytoplasm and be taken up by the ER via a calcium pump, thus replenishing the depleted calcium stores.

Super quick, can happen in about 30 ms

Question 45

how are membrane contact sites important in lipid transfer (what else is also needed to drive the lipid transfer)?

Answer 45

Membrane contact sites are important in non-vesicular transfer of lipids
SER = site of lipid synthesis, then can be distributed to other organelles (through membrane contact sites in unidirectional transfer)

Tethers mentioned earlier at these contact sites, are bi-functional and can act as lipid-transfer proteins

***lipid -transfer proteins
are thought to use concentration gradients as a driver of lipid transport (high lipid conc in ER etc…)
Lipids need to protected from aqueous environment - so transfer of lipids without membrane fusion require the lipid-transfer proteins to have a hydrophobic core

Question 46

Niemman Pick disease C is a defect in lipid transfer proteins - what does it cause?

Answer 46

Cholesterol is delivered to lysosomes, then need to be transferred out to other organelles
A certain one of these proteins normally transfers cholesterol to other organelles. Defect results in an accumulation of cholesterol in lysosomes

Question 47

how are membrane contact sites used in cell signalling?

Answer 47

ER has very close contacts with early and late endosomes in the endocytic pathway

Epidermal growth factor receptor - EGFR (an RTK) is phosphorylated when activated at plasma membrane

When activated, it moves through endocytic pathway to lysosomes for degradation

It must be dephosphorylated by a PTP - protein tyrosine phosphatase localised on the ER. This dephosphorylation and therefore deactivation occurs at an ER-endosome membrane contact site. Once deactivated the EGFR can continue on to the lysosome to be degraded

Question 48

how are membrane contact sites important in organelle fission

Answer 48

ER tubules wrap around the organelle, the ER membrane will have proteins to help promote organelle fission
Mitochondria constantly undergo fission and fusion, live cell imaging has shown how ER tubules can encircle mitochondria to encourage fission

Question 49

what are two diseases linked to membrane contact sites?

Answer 49

Charcot-Marie-Tooth disease can also have mutations in Rab7 that affect lysosome-mitochondria contact sites

Niemman Pick disease C is a defect in lipid transfer proteins