Section 5: Golgi

Question 1

Describe the 3 types of cisternae and the 2 types of tubules in the golgi complex

What is the function of the golgi?

Answer 1

• 3 types of cisternae:
  
  1. Cis: closest to the ER
  2. Medial: in between
  3. Trans: closest to the cell surface
  • Vesicles at cisterna tips bud off to move from one to another
• 2 flanked networks of tubules:
  
  1. CGN (cis golgi network): closest to the ER
  2. TGN (trans golgi network): farthest from the ER

Function: process and sort proteins from the ER

Question 2

Describe the process of budding and fusion and the proteins required (coat, snare, etc.)

Answer 2

Budding = forming a vesicle

• Requires coat proteins
  
  • COPI: for CGN to RER (retrograde transport: toward the nucleus)
  • COPII: for RER to cis golgi (anterograde transport: away from the nucleus)
    
    • COPI and COPII are Sec proteins
  • Clathrin: for TGN to late endosomes and lysosomes (“sideways” transport)

Fusion = fusing with the target membrane

• Requires snare proteins
  
  • Recruited by Sec proteins
    
    • Cannot interact until they are removed from the vesicle
  • Fusion requires ATP

Question 3

What is the purpose of GTP during vesicle transport?

Answer 3

• GTP bound proteins recruit Sec proteins (in the CGN for COPI or RER for COPII)
• GTP proteins are hydrolysed to remove the coat proteins and expose the snare proteins for fusion

Question 4

How does cargo get from the RER to the CGN?

What happens to the vesicle after use?

Answer 4

Cargo must be recognised by COPII

• Cargo can be soluble (in lumen/”outside of cell”) or membrane bound
  
  • Membrane cargo have their ligand binding domain in the lumen
  • Membrane cargo have an amino acid motif called DXE sorting signal (Asp-X-Glu) recognised by COPII
  • Soluble cargo is also recognised by COPII but the mechanism is not known

Vesicles are recycled

1. GTP proteins recruit COPI
2. COPI reforms the vesicle and recruits snare proteins
3. GTP is hydrolysed, coat proteins come off, and snare proteins can bind and refuse using ATP yay!

Question 5

What is cystic fibrosis and what goes wrong?

Answer 5

CF is a recessive genetic disease characterised by abnormal transport of chloride across epithelium resulting in inappropriate hydration causing mucous secretions from cells

• Affects the lung, liver, pancrease, intestines
• Caused by a mutation in the gene for the protein cystic fibrosis transmembrane conductance regulator (CFTR)
  
  • The mutation is usually ΔF508 which affects the ability of CFTR to bind COPII so it is stuck in the ER
  • ΔF508 mutation interferes with the DXE/diacidic sorting signal by deleting one of the amino acids on the CFTR

Question 6

I accidentally transported my RER protein (BiP, calnexin, calreticulin, etc.) to the CGN! How do I get it back?

Answer 6

• ER-resident proteins have a KDEL sorting signal (Lys-Asp-Glu-Leu)
• The KDEL receptor contains C-terminal KKXX sequence and is pH sensitive
  
  • When in the CGN which has a low pH, it will bind to KDEL and COPI and be taken back

Question 7

Describe glycosylation in the golgi

Answer 7

• 3 compartments to the golgi (cis golgi, medial gogli, trans golgi) all have unique glycosylation functions and enzymes
  
  • As proteins bud from different levels, they will differ in characteristics due to the different enzymes they were exposed to
• 2 main types of glycosylation enzymes:
  
  • Glycosidase: removes sugars
  • Glycosyltransferase: adds sugars

Question 8

After moving to the golgi, what 5 things can happen?

Answer 8

Most trafficking occurs from the trans-golgi network

1. Lumen proteins bud off and move to the plasma membrane
   
   • Unknown mechanism
2. Transmembrane proteins bud off and sit on the plasma membrane during fusion
   
   • Unknown mechanism
3. TGN to the trans-golgi
   
   • COPI is used (retrograde)
4. TGN to the lysosome
   
   • Rare
   • Use AP (adaptor protein) coated vesicles
5. TGN to the late endosome
   
   • Common, can take the proteins to many areas
   • Clathrin is used (sideways)

Question 9

Describe the process of TGN –> lysosome transport

Answer 9

Enzymes that degrade fats move to the lysosome, which functions to degrade fats (lol)

Lysosome-targeting sequence: M6P (mannose-6-phosphate), a carbohydrate residue

Added to lysosome destined cargo in the cis golgi via phosphorylation

Question 10

Describe the process of TGN –> late endosome transport

Answer 10

• Most lysosomal targeted proteins first pass through the late endosome
  
  • These can come from the cell surface (receptor-mediated endocytosis) or directly from the TGM
    
    • M6P at the cell surface is recognised by AP2 and the M6P receptor
    • M6P in the TGM is recognised by AP1 and the M6P receptor
• Vesicle formation involves clathrin
  
  • After transport, the clathrin is lost and the M6P releases due to the low pH in the endosome
    
    • M6P receptors return to the TGN or the cell surface

Question 11

Describe low-density lipoproteins (LDLs)

Answer 11

• Lipids (ex., cholesterol) are transported in large particles called lipoproteins (amphipathic shell, apolar core for protecting hydrophobic groups)
• LDL receptors are concentrated in clathrin/AP2 pits (2% of cell surface)
• 2 components to the LDLR:
  
  1. Short C-term NPXY amino acid segment
     
     • Faces the inside of the cell and recognises AP2 for coating
  2. Long N-term ligand binding & b-propeller domain
     
     • At cell surface pH, ligand binding domain binds to ApoB
     • At acidic/endosome pH, histidine residues in the b-propeller domain are protonated, releases the LDL inside the endosome (the LDL receptor recycles to the cell surface)

Question 12

How are endosomes/lysosomes adicified?

Answer 12

• V-class proton pumps transport H+ across membranes using ATP
  
  • Anions passively follow protons resulting in acidification of lumens
• Cl- channels are also present

Question 13

Describe the importance of internalising LDL

Answer 13

• If cholesterol remains in the blood and never moves to the lysosome, it forms plaques
• Ex., familial hypercholesterolemia
  
  • Caused by LDLR mutations such as:
    
    • No LDL receptor
    • Receptor binds LDL poorly
    • Receptor can’t internalise LDL
  • Results in heart attacks and stroke

Question 14

Describe the transferrin cycle

Answer 14

• Iron + transferrin protein = ferrotransferrin (not bound = apotransferrin
  
  • Ferrotransferrin can bind to the transferrin receptor at the cell surface
• As pH drops in the endosome, the iron is released from the ligand and apotransferrin is recycled
  
  • It is released at the cell surface (neutral pH)

Question 15

Describe autophagy (self eating)

Answer 15

• Something things go wrong and the cell needs to remove large components of itself (ex., nonfunctioning mitochondria)
  
  • Autophagy surrounds the item to be degraded in a double-membraned autophagic vesicle
    
    • Involves Atg proteins that are lost with age