ER --> Golgi

Question 1

overview

Answer 1

• proteins that have entered the ER are destined from the Golgi complex are packed in COPII coated vesiciles
• some cargo proteins are actively recruited into vesicles - where they become concentrated
• cargo proteins display exit signals and are recognised by complementary receptor proteins

Question 2

How are proteins transported from the ER to the Golgi?

Answer 2

• by binding to the COPII coat, membrane & cargo proteins are concentrated in transport vesicles that leave the ER.
• cargo proteins are packaged into the vesicle through interactions of exit signal on their cargo receptors
• the exit signal portion of the cargo receptor is embedded in the COPII coat

Question 3

What happens to proteins that are not folded correctly

Answer 3

• Only proteins that are properly folded can leave the ER (& multimeric protein complexes must be completely assembled.)
• those misfolded/incomplete are retained by chaperones, e.g. BIP
• chaperones cover the exit signal/anchor the proteins to the ER
• these proteins are transported to the cytosol for degradation by proteosomes.

Question 4

What happens after the COPPII coated vesicles has budded of the ER?

Answer 4

• COPPII coat is shed, energy from this is derived from ATP hydrolysis
• vesicles begin to fuse to one another

Question 5

How does fusion of two vesicles take place?

Answer 5

• requires matching SNARES, on adjacent identical membranes
• different snares bind with each other, v-SNARE bind with t-SNARE
  -zipping up from the amino termini and drawing the two membranes together
• zipping causes curvature and lateral tension on bilayers, favouring hemifusion between outer and inner leaflets
  and causing formation of an energetically unfavourable void space.
• inner leaflets of both membranes come into contact
• pore widens - vesicle contents are released

Question 6

What does fusion require?

Answer 6

• cells recognise each other (through V & T snares)
• there surfaces become closely apposed, which requires removal of water normally associated with polar head groups of lipids
• their bilayers become locally disrupted, resulting in fusion of the outer leaflet of the bilayer (hemifusion)
• bilayers fuse to form a single continuous bilayer
  –> fusion is triggered at the appropriate time or in response to a specific signal
  -

Question 7

What is Caveolin?

Answer 7

• caveolin is an integral membrane, with two gobular domains, connected hairpin-shaped hydrophobic domain (hairpin binds the protein to the cytoplasmic leaflet of the plasma membrane)
• caveolins oligomerise - oligomerization leads to formation of caveolin-rich microdomains in the plasma membrane (cytosol side); it’s necessary for formation of caveolar endocytic vesicles.
• caveolin binds cholesterol in the membrane, and the presence of caveolin forces the lipid bilayer to curve inward, forming caveolae (‘little caves’)
• fission of the vesicle from the plasma membrane is mediated by GTPase dynamin II, which is localised around the neck of the budding vesicle.
• the released caveolar vesicle can fuse with early endosome or caveosome, transcytosed?
• do not shed caveolae coat

Question 8

Roles of caveolae

Answer 8

• transcytosis of albumin in edothelial cells
• internalization of insulin receptor in primary adipocytes
• caveolae can be used for entry to the cell by some pathogens to avoid degradation in lysosomes.

Question 9

What are some of the functions of endocytosis?

Answer 9

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Question 10

What are the fates of receptor-mediated endocytosed proetins?

Answer 10

• proteins recycles from endosomes to plasma membrane, e.g. receptors
• degraded by lysosome
• moved to a different part of plasma membrane (transyctosis).

Question 11

Discuss an example of receptor-mediated endocytosis - cholesterol

Answer 11

• most cholester moved in blood as lipid-protein
• ‘low-density lipoproteins’ (LDLs)
• if a cell needs cholester, it synthesises more LDL cholesterol receptors and inserts them into the plasma membrane
• LDL particles bind
• LDL receptor + ligand diffused to form clathrin coated vesicles
• vesicle forms, buds off and moves inside the cell
• clathrin coat shed
• LDL receptor + ligand delivered to endosome
• low pH in endomsome causes receptor to release LDL ligand and fuse with lysosomes
• cholesteryl esters are hydrolysed to give free cholesterol for the cell, receptors are recycled.

Question 12

Clinical relevance of cholesterol receptor mediated endocytosis

Answer 12

• LDL receptor defective, missing or can’t bind to LDL or clathrin
• cells can’t take up cholesterol
• too high blood cholesterol (not enough cholesterol in cells), leads to increased risk of coronary heart disease).

Question 13

Discuss an example of receptor-mediated endocytosis - iron uptake

Answer 13

• Transferrin + iron binds to receptor on cell
• receptor is endocytosed, etc
• vesicle fuses with endosome, low pH releases iron
• receptor & transferin are recycled
• transfer in released into neutral pH of extracellular fluid, and can pick up more iron

Question 14

What are some of the uses of exocytosis?

Answer 14

• specialised secretory cell secreting hormones, neurotransmitters, digestive enzymes

Question 15

tubular clusters

Answer 15

• fusion of vesicles form tubular clusters
• tubular clusters are short lived as they move along microtubles to the Golgi apparatus
• fuse to golgi and release their contents

Question 16

Tubular clusters

Answer 16

• as soon as tubular clusters form, they begin budding off vesicles of their own
• these vesicles are COPI coated, and carry escaped resident ER proteins back to the ER
• proteins that participated in ER vesicle budding reaction are also returned
• tubular clusters continually mature, gradually changing composition as selected proteins are transport back to the ER

Question 17

Describe the transport retrieval pathway to the ER using sorting signals

Answer 17

• resident ER proteins contain signals that bind to COPI coats
• ER proteins are packaged in COPI coated vesicles
• ER membrane proteins can bind directly to COPI
• BUT ER resident proteins (localised in ER lumen) must bind to ‘KEDL’ receptor.
• ‘KEDL’ receptor is multipass transmembrane protein
• KEDL receptor binds to KEDL signal on ER resident protein (e.g. BiP chaperone protein) and packages the KEDL prtoein into a COPI coated vesicle.
• in order for the KEDL receptor to unload the protein into the ER, the KEDL receptor has high affininity for the KEDL signal on ER resident proteins in vesicles & tubular glusters, but a low affinity for the signal in the ER to release the protein.
• change in affinity depends on change in pH in different compartments. Golgi & tubular clusters have higher pH than ER.

Question 18

How are ER resident proteins retain in the ER?

Answer 18

• the ER proteins bind to one another, forming complexes too big to enter transport vesicles
• only proteins that escape retention are returned via the KEDL pathway.
• aggregation of proteins that function in same compartment ‘kin recognition’

Question 19

How does length of membrane protein determine its location?

Answer 19

• vesicles that leave golgi destined for the plasma membrane are rich in cholesterol
• cholesterol fills the space between kinked unsaturated hydrocarbons - causing tighter horizontal alignment, but increasing separation between lipid head groups of the two leaflets of the bilayer (increasing width of plasma membrane)
• the plasma membrane is thicker than the goligi membrane
• transmembrane proteins must be sufficiently long transmembrane segments to span the plasma membrane thickness - if they are to enter cholesterol rich transport vesicle destined for plasma membrane