MODULE 6: protein trafficking

Question 1

COP-II dependent vesicular transport (5 stages)

Answer 1

STAGE 1: PRIMING

• sec12 (nuclear exchange factor) recognises inactive form of sar1 (GDP, sar1 = GTPase)
• sec12 recruits and activates sar1 (GTP)
• activated sar1 undergoes conformational change such that hydrophobic N-terminus associates with membrane (this defines specificity of where process will occur)

STAGE 2: COAT ASSEMBLY

• active sar1-GTP is bound to ER membrane
• recruits Sec23/24 subcomplex
• sec23/24 captures different cargo types
• sec13/31 recuited to stabilise polymerisation and budding
• COP-II vesicle pinches off and is released from membrane

STAGE 3: COAT DISASSEMBLY

• ** coat is recycled
• ** dense coat prevents recognition by other machinery
• ** coat is stable and requires active process to be removed
• sec23 promotes hydrolysis GTP on sar1 to GDP
• energy from hydrolysis causes conformational change that uncouples sar1-GDP from vesicle and disassembly of coat
• uncoated vesicle exposes membrane protein tails

STAGE 4: TRANSLOCATION + DOCKING

• kinesin moves vesicles towards plus end
• dyactin moves vesicles owards minus end
• vesicle is tethered via SNARE proteins to target membrane —> close proximity
• rab-GTP anchored to the vesicle engages a rab effector on the acceptor membrane

STAGE 5: FUSION

• NSF and SNAP molecules detect cis-SNARE complexes and use ATP to dissasemble them
• causes fusion of vesicle and membrane
• following fusion, NSF and a-SNAP bind to the SNARE complex for recycling

Question 2

protein sorting mechanisms

Answer 2

segregation:

• different cellular regions have different biochemistry (e.g. pH)
• machinery segregated according to biochemistry
• KDEL motif occurs on ER proteins
• binds in cis=Golgi network but not in rough ER —> even though receptor is in both organelles
• pH and post-translational modification prevent binding of KDEL to receptor in rough ER —> selective function

retrieval:

• to concentrate cargo, no active exclusion of transport out, but actively transported back to target organelle
• retrieval results in concentrated protein in organelle
• V-SNARES retrieved in this way

Question 3

transport pathways within trans-golgi network (3)

Answer 3

3 options once molecules reach TGN

1. constitutive:
   - no protein signal
   - protein packaged and non-selectively secreted
   - on going pathway
   - albumin,
2. regulated
   - molecules selectively segregated
   - incoorperated into secretory vesicles
   - release of vesicles is controlled
3. sorting into lysosomes
   - destined for lysosomal system
   - become part of soluble proteins which make up lysosomes

Question 4

TGN to lysosome pathway

Answer 4

1. newly synthesised precursors recognised by enzymes as it moves through the golgi
2. modified —> phosphorylated on 6th position
3. modified proteins recognised by M6P receptor
4. ligand binds to receptors
5. recognised by clathrin coat and vesicle forms
6. transport to lysosome + uncoating
7. fusion with lysosome
8. uncoupling and activation of precursor
9. receptor incoorperated into retrograde transport —> back to TGN

Question 5

receptor-mediated endocytosis via clathrin-coated pits

Answer 5

for example: LDL (low density lipioprotein)

• N-terminus of LDLR binds apoprotein B in LDL
• —–> at pH=5, LDL particle disassociates from receptor and is trafficked back to cell surface
• —–> at pH=7, LDL binds to receptor
• C-terminus of LDLR binds adaptin complex
• sorting signal from cytoplasm recognised by adaptin complex
• clathrin-coat begins to assemble and vesicle forms
• vesicle pinches off, facilitated by dynamin
• —–> dynamin is a GTPase —> hydrolyses GTP to GDP + P(i) + energy
• —–> two dynamin rings twist to provide mechanical force to pinch off vesicle