33- Post-Golgi protein trafficking Flashcards
Constitutive secretion (4)
transport of protein from TGN to PM
- Operates continuously in call cells
- Proteins without sorting signals can enter pathway non-selectively
- Cargoes are released without external stimuli
-Small amount - Housekeeping pathway
-Newly synthesized PM proteins & lipids in ER
–Must deliver to PM (extracellular space) to grow cells & communicate with other cells
Regulated secretion (4)
transport of protein from TGN to PM
- Operates only in some cells
- Proteins with proper sorting signals are selectively packaged into secretory vesicles in TGN & often further matured/concentrated on the way to PM
- Cargoes only released when cells stimulated to do so
- Used to secrete products on high demand (not often)
-Insulin secretion in pancreatic cells
-Neurotransmitter in neurons
Maturation & concentration of insulin in secretory vesicles:
Proinsulin
3 domains with disulfide bond (B-C-A)
Proinsulin Ab: away from TGN – densely packaged
-In vesicles near TGN – loosely packaged
Maturation & concentration of insulin in secretory vesicles:
Insulin
2 domains with disulfide bond(B-A)
Insulin Ab: Densely packaged insulin away from TGN
-Less near TGN, but still some present
Insulin secretion occurs
rapidly when needed
After insulin molecules are matured & concentrated in secretory vesicles
They are docked at PM of pancreastic cells = Wait for signal
Trans-SNARE only formed but not tightly wrapped around
If blood sugar level is below threshold:
Insulin condensed secretory vesicles wont fuse with PM = no release of insulin
No tight wrap-around of trans SNARE
When blood sugar increases to a threshold level:
Insulin condensed secretory vesicles rapidly fuse to PM= Large # of insulin is released into blood stream in short time
Tight wrap-around of trans-SNARE
2 unsolved key issues in protein secretion to cell surface
Biochemical identity of vesicles involved in protein secretion to cell surface = not know
-Because it is difficult to purify
Sorting signals involved in selectively protein packaging in secretory vesicles in trans-Golgi network in regulated secretion = not known
-Ex: sorting signal in cargo
Protein trafficking from TGN to lysosome (side branch transport pathway):
protein involved (2)
Clathrin & AP1 to make a type of vesicle to transport lysosome enzymes to lysosome via late endoscope
Clathrin: triskelion-shaped proteins
-3H chain
-3L chain
Protein trafficking from TGN to lysosome pathway: step by step
Assembly & pinching off of CCVs
- Formation of clathrin coated vesicles (CCVs) require polymerization of clathrins & AP (adaptor protein) on membrane surface of either TGN or PM
- In CCVs formed from TGN, AP1 interacts with integral membrane cargoes and/or cargo receptor proteins
-So cargoes are actively packaged into CCVs - In CCV formed from PM, AP2 select the cargoes
-Pick up cargo from PM - For CCV pinching off – Dynamin (large GTPase) undergo GTP-dependent polymerization over CCV neck to squeeze vesicles & GTP hydrolysis = release
Long necked clathrin bud forms but can’t pinch off if…
in presence of non-hydrolysable GTP𝛾S in in vitro experiment
-End up with transport problem
-GTP𝛾S always active in dynamin
If can’t be hydrolyzed = cant pinch off
If no active GTP =
- No long neck forms
- No polymerization around neck of CCV
- Protein transport issue
- Inactive dynamin
sorting signal allowing lysosomal enzymes to be packaged into CCVs
M6P
Trafficking of lysosomal enzymes
M6P
-To be packaged in COPI/II
-All lysosomal enzymes possess sequence patch
-The sequence is not seen on proteins going to extracellular space
Adding M6P to glycan of lysosomal enzymes
- When glycosylated lysosomal enzyme enters cis-Golgi
-GlcNAc phosphotransferase recognizes it & adds a phosphorylated GlcNAc group to terminal mannose - Modified protein released from GlcNAc phosphotransferase & passes to trans-Golgi at where phosphodiesterase removed GlcNAc group
-Leaves a phosphate at terminal mannose
M6P receptor bind to M6P signal = … dependent
pH
At 5.0 = release cargo
Trafficking of lysosomal enzymes to lysosomes, what kind of transportation?
Constitutive
transport in small # to PM/extracellular space
Trafficking of lysosomal enzymes to lysosomes: step by step
- Clathrin coated bud with M6P receptor & M6P signal
-Clathrin triskelion+AP1 complex
-TGN : pH ~6.5 - Clathrin coated vesicles
-Dynamin cleaves of CCV - Uncoated transport vesicle fuse with Late endosome (low pH)
-M6P release cargo - M6P receptor recycled back to TGN or PM
- Delivery to lysosome
-Functional active enzyme
-Phosphate group removed
to take Escaped lysosomal enzyme back to cells, we use…
Receptor-mediated endocytosis
Cell growth – constitutive secretion need to be
balanced by endocytosis
don’t want giant cells
Take nutrient into cell (cholesterol)
-Take cholesterol from LDL to cells
Downregulates EGF at times
Cell uptake all material by endocytosis, mediated by…
Clathrin AP2
what is LDL
LDL – low density lipoprotein
-Bad but needed it – need to take efficiently
Need cholesterol from LDL to make healthy cells
-Phospholipid monolayer
-Cholesteryl ester –attached to fatty acids
LDL receptor-mediated endocytosis of LDL: step by step
- Binding of LDL to LDL receptor in extracellular space
-pH dependent (~7.0) - Polymerization of clathrin AP2
- Uncoating to make early endosome
-Fusion of uncoated vesicles - early endosome Fuse to late endosome
- LDL released from LDLR in late endosome
-pH = 5.0 = binding of LDLR and LDL - LDL delivered to lysosome & digested to release cholesterol
- LDL receptor recycled back to PM for reuse
Human LDL receptor (LDL-R) & LDL: pH dependent
pH~7.0 – LDL receptor:
-β-propeller domain
-Sorting signal between receptor & AP2
pH~5 – endosome
-surface of β-propeller domain becomes + & binds to ligand binding arm
-released LDL particle
