Chapter 17

Vesicular Traffics, Secretion, and Endocytosis

SNARE : SNAP Receptor

SNAP : Soluble NSF Attached Protein

NSF : N-ethylmaleimide (NEM)- Sensitive Factor

COP I : Coat Complex I (Coat Protein)

COP II : Coat Complex II

GGAs : Golgi-associated γ-Adaptin homologues,

Arf-binding

ARF : ADP-ribosylating Factor

VAMP : vesicle-associated membrane protein ( v-SNARE)

Main Content:

1. Techniques for studying the secretory pathway

2. Molecular mechanisms of vesicular traffic

3. Early stages of the secretory pathway

4. Later stages of the secretory pathway

5. Receptor-mediated endocytosis and the sorting of

the internalized proteins

6. Synaptic vesicle function and formation

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Proteins are first transported from ER to Trans-Golgi Network (TGN), and then, transported further via three separate pathways:

1. Constitutive secretion : to the plasma membrane

2. Regulated secretion : stored as secretory vesicle, and

released with stimulus

3. From TGN to late endosomes and then lysosomes

Molecular Mechanisms of Vesicular Traffic

Donor

Vesicle Formation

Budding

Docking

FusionWith Acceptor

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Coat Assembly and Disassembly

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How do vesicles select /recruit correct cargo proteins?

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Docking and Fusion of Transport

Vesicle with its Target Membrane

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(Summary)Molecular mechanisms of vesicular traffic

1. Three types of transport vesicles : COP I, COP II and clatherin

2. Polymerization of cytosolic coat proteins to form a coat for the vesicle

3. Pinch off & release vesicle, decoating→move →docking→fusion

4. GTP- binding proteins (Sar-1, ARF):Control: coat formation; vesicular budding; decoating

5. Select proper proteins for the vesicles6. GTPase (Rab) : control docking7. SNARE, SNAP complexes initiate fusion8. NSF (ATPase) initiates SNARE complex disassembly

Early stages of the secretory pathway

ER→Cis-Golgi : Anterograde

Cis-Golgi→ER : Retrograde

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3 components for COP II :

1) small GTP binding protein, Sar-1

2) a Sec 23 / Sec 24 complex

3) a Sec 13 / Sec 31 complex

Sorting signals for COP I vesicles:

Membrane : KKXX (Lys, Lys, XX)

ER soluble : KDEL (Lys, Asp, Glu, Leu )

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Vesicle traffic in the early stages of the secretory pathway (summary)

1. COP II vesicle : protein transport ER→Cis-Golgi

2. COP I vesicles : from Cis-Golgi → ER ; from later to earlier compartment in Golgi stack

3. COP II: containing three components

4. COP II : sorting signal for membrane cargo proteins : Di-acidic (Asp-X-Glu)

5. Sorting signal for membrane binding to COP I is KKXX (Lys, Lys, XX)

Vesicle traffic in the early stages of the secretorypathway (summary)

6. Sorting signal for soluble ER protein containing in

COP I is KDEL ( Lys, Asp, Glu, Leu)

7. Fundamental features of this protein trafficking:

a) selective entry of proteins into suitable vesicle

b) recycling of membrane phospholipid and proteins, and soluble luminal proteins between the two compartments

8. Cisternal progression: from cis- , medial, to trans-Golginet work (TGN)

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Later stages of the secretory pathway

From TGN:

1) Secretion (? Vesicle coat)

2) To plasma membrane (? Vesicle coat)

3) To late endosomes, then lysosomes

(clathrin / AP)

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Types of clathrin/AP coat:

1. Clathrin / AP 1 or GGA: recognize TyrXXфfrom TGN→ late endosome (LE)→lysosome

2. Clathrin / AP2 : recognize TyrXXф (YXXф)from plasma membrane during endocytosis

3. Clathrin / AP3from TGN to lysosome directly or deliver proteins to melanosome containing the black pigment melanine in skin cells

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Decoating

Clathrin/AP, COPI, COPII vesicles→

Pinch off→ lose their coat

Possible mechanism: Some chaperone proteins in cytosol,

such as Hsc 70 will depolymerize the clathrin by using

the energy released from ATP hydrolysis.

Lose coat:

1. reuse triskelion

2. expose V-SNARE for docking and fusion

3. As ARF are switched from the GTP-bound to GDP-

bound, it is the time for clathrin coat to be depolymerized

Mannose-6 PhosphateSorting Signal for Lysosomal Enzymes

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Secretory Pathway1. Constitutive secretion : constitutive vesicles

2. Regulated secretion : secretory vesicles

The coat proteins for either constitutive or regulated

vesicles are unknown

Regulated Secretion:

1. trans-Golgi network → bud out→store in a special vesicle, and release by a special stimulus

2. A common mechanism appears to sort regulated proteins. These proteins selectively aggregate together , and withchromogranin A, chromogranin B and secretogranin II before their incorporation into the vesicles.

Proteolytical Processing ofsecretory proteins

Proproteins→ Proteins

Occurs after the secretoryvesicles budded from the TGN

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Receptor-Mediated Endocytosis and

the Sorting of Internalized Proteins

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Type II Familial Hypercholesterolemia

1.normal binding between LDL and LDL receptor, but, no internalization

causes: no interaction between LDL receptor and

clathrin/AP2 coated pits, due to the lack of

sorting sequence

NPXY (Asn, Pro, X, Tyr)

2. In some patients: LDL receptor is normal, but AP2 gene defect, which cannot recognize NPXY

Sorting signals of receptor targeting

into clathrin/AP2 coated pit:

1. NPXY (Asn, Pro, X, Tyr)

2.YXXΦ (Tyr, X, X, bulky hydrophobic amino acid)

3.Leu-Leu

4.ubiquitin binding→endocytosis

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