1. Lecture 12: The secretory pathway
The fission and fusion of transport carriers
Protein sorting in the secretory pathway
Recommended reading: MCB (Lodish), pages
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2. How are proteins transported out of the ER
and along the secretory pathway?
Vesicle formation: How to select cargo proteins?
How to induce membrane curvature?
How to induce membrane fission?
Vesicle fusion: How to choose to the correct target membrane?
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3. How is vesicle formation initiated?
Recruitment and activation of a small GTPase to the donor membrane is the first step of vesicle formation.
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4. How are vesicles formed?3. 2. 1.
1. Recruitment of a small GTPase to the donor membrane (mediated by its GEF)
small GTPase is in its GTP-bound form
2. Activated small GTPase mediates the recruitment of coat proteins
3. Coat subunits selectively bind and recruit cargo into vesicle
coat assembly leads to - cargo recruitment
- membrane curvature.
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5. How do vesicles look by EM?
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7. V-SNARE
t-SNARE
The fusion of a vesicle requires the formation of the v-SNARE/t-SNARE complex
on the vesicle: v-SNARE helix
on the target membrane: t-SNAREs: 1 helix from actual t-SNARE
2 helices from SNAP-25
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10. Bacterial toxins disrupt vesicle fusion
Toxins of Clostridium botulinum and Clostridium tetani prevent neurotransmitter release by cleaving the membrane fusion machinery
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11. A cycle of vesicle budding and fusion Sorting information:
Membrane proteins
sorting signal is contained in the protein
Soluble proteins
Soluble cargo protein binds to a transmembrane receptor that contains a sorting signal.
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12. Vesicles of the secretory pathway
COPI
Clathrin ?
COPII
Anterograde (Forward) transport (red arrows)
Retrograde (backward) transport (blue arrows)
Endocytic transport (green arrows)
Reminder: Mitochondria and peroxisomes are not part of the secretory pathway
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13. Protein transport can be visualized by live-imaging.
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14. What happens to proteins that have “escaped” the ER?
Retrieval signals:
Proteins that predominantly function in the ER (such as BiP) contain retrieval signals (eg KDEL)
recognized by a receptor
mediate retrieval of escaped protein.
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15. Golgi apparatus consists of stacked cisternae
Do Golgi cisternae differ functionally? If so, how can we tell?
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16. Processing of N-linked oligosaccharides in the Golgi
Endo H sensitive
Endo H resistant
Each Golgi cisternae contains a unique set of glycosylation enzymes that modify the N-glycan on a secretory protein as it is transport across the Golgi stack.
Enzymes of the glycosylation machinery can be used as cisternae-specific markers.
Changes of the N-glycan of secretory proteins are used to monitor protein transport.
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17. How is large cargo transported?
50 to 70 nm
400 nm
300 nm
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18. How are large molecules secreted?
Example: collagen
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19. How are proteins transported across the stack of Golgi cisternae?
anterograde and retrograde transport between the cisternae is mediated by transport vesicles (COPI)
anterograde transport is mediated by forward movement of the entire cisternae. Golgi resident proteins are recycled by transport in COPI-coated vesicles.
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20. What are the options of a protein in the TGN?
Nucleus ER
Golgi
Endosome/Lysosome
Plasma membrane 1 2 3 4
Stay in the Golgi
regulated secretion: sorting into storage granules (eg Neurotransmitters)
Constitutive secretion
Sorting to the endosome
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21. Constitutive (2) and regulated (3) secretion
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22. Sorting of proteins into storage granules
Before signal
after signal
Mast cell secreting histamine
What is stored? Hormones, mucus, neurotransmitters
“Sorting signal”: proteins selectively aggregate with one another in TGN
Aggregation allow 200 fold concentration in storage granules
Sorting machinery: not known
Exocytosis is triggered by a signal (often chemical signal)
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23. Lysosomes
300/cell
Membrane-bound acidic compartment (pH5 is actively maintained)
major site of intracellular digestion (40 types of hydrolytic enzymes that are optimally active in acidic conditions and that are heavily glycosylated)
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24. How are proteins sorted to the endosome/lysosome?
pH 6.5 to 6.7
pH 6
The interaction between Mannose-6-phosphate and its receptor is regulated by pH.
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25. Important concepts, lecture 12
Vesicle formation (summary):
initiated by membrane bound GEFs that recruit and activate small GTPases. small GTPase-GTP recruits coat proteins from the cytosol which then recruit cargo (via specific receptors) and which also promote membrane curvature. Upon budding, GTP on the small GTPase is hydrolyzed leading to the vesicle uncoating and exposure of the fusion machinery. While the mechanism of vesicle formation is conserved between vesicles of different transport steps, the proteins that make up the vesicles are NOT conserved.
Vesicle fusion (summary):
Vesicle fusion depends on the presence of v- and t-SNAREs which form a bundle of 4 helices --> promote fusion by bringing membranes in proximity. The bundle of helices is disassembled by the ATPase NSF and accessory proteins. v-SNAREs are recycled to their site of origin.
v-and t-SNARE pairing and specific rab proteins (small GTPases) determine the target membrane.
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26. Lecture 12: Important concepts (2)
ER exit requires sorting signals and is mediated by COPII-coated vesicles.
missorted ER proteins are retrieved by a signal- and receptor- dependent retrieval mechanism (mediated by COP I-coated vesicles)
Golgi membranes are organized as ordered series of functionally distinct compartments.
As proteins are transported across the Golgi cisternae, their N-linked glycans are modified by specifically-localized glycosyltransferases and glycosidases, which serve as marker proteins
Protein transport across the Golgi is believed to occur by cisternal maturation
protein sorting at the TGN: While sorting signals for non-regulated secretion remain to be identified, sorting signals are required for staying in the Golgi, being transported to the endosome/lysosome (M6P) or for being included in storage granules (that are exocytosed in response to a signal (eg Ca2+ for neurotransmitters, regulated aggregation).
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