Vesicular traffic, Secretion and
Endocytosis
e
x
Compartmentalization creates potential
problems for cells
How are proteins (transcription factors, DNA polymerases,
etc.) after synthesized in the cytoplasm are delivered to
nucleus to perform their physiological functions?
Nuclear Pore Complex Mediated Gated Transport
Compartmentalization creates potential
problems for cells
How are proteins (growth factors, insulins, hormones, etc)
after synthesized in the cytoplasm are delivered from inside
of the cell to the outside of the cell to perform their
physiological functions?
Secretory transport pathway
How molecules (nutrients, cholesterol, ions) are delivered from
outside of the cell to inside of the cell?
Endocytic transport pathway
Outline
Lecture I
• General introduction
- Transport vesicles
- Roadmap of secretory transport pathway
• Principles underlying vesicular transport
Lecture II
• Secretory transport pathway
• Endocytic transport pathway
Secretory and endocytic transport pathways are
mediated by vesicular carriers
Transport vesicles
Transport vesicles:
- small spherical shaped
- membrane enclosed
- enriched with cargo proteins
Vesicular transport is fundamentally important
胰臟腺泡腺細胞
In a neuron, synaptic vesicles store various
neurotransmitters that are released at the
synapse
Pancreatic acinar cell secretes digestive
enzymes from zymogen granules (ZG)
The road-map of the secretory pathway
George Palade
Father of modern cell biology
Nobel Prize
Physiology and Medicine, 1974
ER
Golgi
Plasma membrane
The road-map of the secretory pathway
George Palade
Father of modern cell biology
Nobel Prize
Physiology and Medicine, 1974
ER
Golgi
Plasma membrane
Direct visualization of trafficking of cargo
proteins along secretory transport pathway
Fluorescent tag
How to identify the genes and proteins that are involved in each step
of the secretory transport pathway?
ER
Golgi
Plasma membrane
Genetic approaches to reveal genes that
regulate vesicular trafficking
2013 Nobel Prize in
Physiology or
Medicine
Wild type
Sec1 mutant
Separation of wild type and mutant yeast cells
through density gradient separation
Wild type and Sec1 mutant
yeast cells in 100:1 ratio
are mixed and distributed
throughout the density gradeint
Low density
yeast cells
(wt)
Centrifugation
High density
yeast cells
(Sec1 mutant)
Mutations that block protein / membrane traffic
at distinct step in yeast
Mutations that block protein / membrane traffic
at distinct step in yeast
Summary I
• General introduction
- Transport vesicles are fundamentally important for
various physiological processes
- Roadmap of secretory transport pathway
ER
Golgi
Plasma membrane
- Yeast genetic study to identify genes and proteins that
are important for distinct steps in the secretory transport
pathway
Outline
Lecture I
• General introduction
- Transport vesicles
- Roadmap of secretory transport pathway
• Principles underlying vesicular transport
Lecture II
• Secretory transport pathway
• Endocytic transport pathway
Essential steps of vesicular transport
1. Vesicle budding and cargo protein
packaging
How lipid bilayers are deformed?
How cargo molecules are selected?
How vesicles are pinched off from the
donor compartment?
2. Vesicle transport
How vesicles are delivered from one
place to the other place?
3. Vesicle fusion with its target organelle
How vesicles are targeted to
their correct destinations?
How lipid bilayers are fused?
Vesicle budding is driven by protein coats
Different coats mediate different
trafficking steps
The assembly and disassembly of a
clathrin coat
- Cargo proteins recognized by cargo receptor
- Cargo receptor recruits adaptin
- Adaptin recruits clathrin
- Polymerization of clathrin forms clathrin-coated
membrane patch then clathrin-coated pit
- Clathrin-coated pit pinches off
the plasma membrane
- Clathrin coat is released from vesicles
Clathrin-coated vesicle formation
in action!
Clathrin-coated vesicle formation
clathrin-coated
membrane patch
clathrin-coated pit
Clathrin-coated vesicle formation
How lipid bilayers are deformed?
Polymerization of coat proteins into concave-shaped cage
structures to force the deformation of the lipid bilayer
How cargo molecules are selected?
Specific recognition of cargo molecules by cargo receptors
clathrin-coated
membrane patch
clathrin-coated pit
Clathrin-coated pits
Essential steps of vesicle transport
1. Vesicle budding and cargo protein packaging
How lipid bilayers are deformed?
How cargo molecules are selected?
How vesicles are pinched off from the donor compartment?
2. Vesicle transport
How vesicles are targeted to their correct destinations?
3. Vesicle fusion with its target organelle
How lipid bilayers are fused?
0
Dynamin functions in pinching off
clathrin-coated vesicles
Dynamin functions in pinching off
clathrin-coated vesicles
Mutation in dynamin causes defects in pinching
off clathrin coated pits
Mutations in dynamin causes paralysis at the
high temperature in Drosophila
Drosophila bearing temperature
sensitive mutations in dynamin
Drosophila
wild type
Essential steps of vesicular transport
1. Vesicle budding and cargo protein
packaging
How lipid bilayers are deformed?
How cargo molecules are selected?
How vesicles are pinched off from the
donor compartment?
2. Vesicle transport
How vesicles are delivered from one
place to the other place?
3. Vesicle fusion with its target organelle
How vesicles are targeted to
their correct destinations?
0
How lipid bilayers are fused?
Microtubules are highways in the cell
A highway system for transportation
Vesicles (green) moving on the
microtubule tracks (red)
Splinter et al., MBoC 2012 23:4226
Essential steps of vesicular transport
1. Vesicle budding and cargo protein
packaging
How lipid bilayers are deformed?
How cargo molecules are selected?
How vesicles are pinched off from the
donor compartment?
2. Vesicle transport
How vesicles are delivered from one
place to the other place?
3. Vesicle fusion with its target organelle
How vesicles are targeted to
their correct destinations?
How lipid bilayers are fused?
Rab proteins, tethering proteins and SNAREs regulate
targeting transport vesicles to their target membranes
SNARE proteins catalyze the fusion of vesicle
and target membranes
James Rothman
2013 Nobel Prize in
Physiology or
Medicine
The specificity of SNARE-mediated membrane fusion
15_20_SNAREs.jpg
Summary II:
Essential steps of vesicular transport
Coat proteins (COPI, COPII and clathrin):
deformation of lipid bilayer
packaging cargo proteins into budding vesicles
Dynamin: pinching off vesicles from the donor membranes
Rab proteins, tethering proteins: vesicle targeting
SNAREs: Vesicle fusion with its target organelle
Bonifacino and Glick, 2004