Protein trafficking
from the ER (1)
continued
General mechanisms of protein trafficking
and transport
• How does a vesicle form
from an organelle
membrane?
• How does it select specific
cargo proteins?
Many transport vesicles appear to have
electron dense “coats”
Molecular Biology of the Cell
How does a transport vesicle sort proteins?
Lodish, 7th Edition, page 634
Different types of coats sort proteins
at different transport steps
•The GTPases ARF and Sar1 control coat recruitment.
•They also control disassembly of COPI and II coated vesicles.
•Uncoating of clathrin-coated vesicles requires an Hsp70
family ATPase. (MCB)
Assemby and disassembly of a coated vesicle
(in this case, a clathrin coated vesicle)
After uncoating, vesicles dock and fuse
with the target membrane
t-SNARE on
target membrane
v-SNARE on
vesicle membrane
Specificity of
fusion
Other factors
required
(e.g. tethers,
small GTPases)
Lodish, 7th edition, page 634
Membrane fusion
Molecular Biology of the Cell
Dissociation of SNAREs by NSF after membrane fusion
Molecular Biology of the Cell
Each fusion has different Rabs and different
combinations of SNAREs
Each fusion has different Rabs and different
combinations of SNAREs
v-SNAREs in blue
t-SNAREs in red
and green
(This should not be
taken as a
comprehensive
list)
From: Dingjan I, Linders PT, Verboogen DR, Revelo NH, ter Beest M, van den Bogaart G.
Endosomal and Phagosomal SNAREs. Physiol Rev 98: 1465–1492, 2018.
Phosphoinositides mark organelles and membrane
domains
• Phosphoinositides make up <10%
membrane lipids
• Important regulatory function
• Can be phosphorylated at 3’, 4’, or 5’
position
• Regulated by specific kinases and
phosphatases
• Proteins involved in membrane traffic can
bind specifically to different forms
Molecular
Biology of
PI(3)P PI(4,5)P the Cell
Protein trafficking from the ER (2)
Protein sorting and transport:
examples
Protein sorting and transport: ER resident proteins,
secretion, targeting to lysosomes, endocytosis
Molecular Biology of the Cell
Examples of protein sorting
1. Soluble ER resident proteins
Key:
KDEL
receptor
COPII
soluble ER
protein
ER cis-Golgi with KDEL
eg. BiP
COPI
secreted
protein
Lys-Asp-Glu-Leu (KDEL) at C-terminus
KDEL is a retrieval signal
Examples of protein sorting
1. Transmembrane ER resident proteins
Key:
KDEL
receptor
soluble ER
COPII protein
with KDEL
ER cis-Golgi eg. BiP
secreted
COPI protein
ER membrane
protein
Lys-Lys-X-X (-KKXX) at C-terminus of a membrane protein
-KKXX is a retrieval signal
Key:
plasma Examples of protein
membrane
protein sorting Plasma
membrane
secreted
protein
2. Secretion or exocytosis
hormone
COPII
constitutive
secretion
cis- medial- trans-
ER
Golgi Golgi Golgi signal
regulated
secretion
As a protein travels through the
Golgi stack, it is modified
As a protein travels through the
Golgi stack, it is modified
O-linked glycosylation
•Sugars added to -OH
groups of serine or
threonine
•Catalyzed by a series of
glycosyl transferase
enzymes in the Golgi
Models for the organization of the Golgi and the
transport of proteins from one cisterna to the next
Vesicular transport Cisternal maturation
From: Glick BS and Luini A. Cold Spring Harb Perspect Biol 2011;3:a005215.
Models for Golgi traffic: a critical assessment.
The lysosome
Contains proteases
and lipases for
degrading
proteins and lipids
Can be considered
a “concentrated
bag of enzymes”
Fuses directly with
the late endosome
and is then
reformed
The lumen is acidic
Examples of protein sorting
3. Soluble lysosomal resident proteins
Lysosome
•Mannose 6-phosphate added in the cis-Golgi
•Mucolipidosis Type II or I-cell disease
Molecular Biology of the Cell
Examples of protein sorting
4. Endocytosis of receptors a) LDL receptor
•Receptor-mediated
endocytosis of LDL
Low Density Lipoprotein (cholesterol)
•Signal: -FDNPVY-
How was the LDL receptor
endocytosis signal discovered?
•Joseph Goldstein and Michael Brown were studying
patients with familial hypercholesterolaemia
•One patient had an amino acid change -NPVY- to
-NPVC- in the cytoplasmic domain of the LDL receptor
•LDL still binds the mutant receptor but fails to internalise
•LDL can not be cleared from the blood
Examples of protein sorting
4. Endocytosis of receptors b) EGF receptor
•Receptor-mediated
endocytosis of EGF
(a growth factor)
•Signals: -FYRAL- ,
ubiquitin addition
-YXXØ- is an
endocytosis
Epidermal Growth Factor signal
Ubiquitin is a signal for sorting at late
endosomes (multivesicular bodies)
Molecular Biology of the Cell
ESCRTs mediate cytokinesis, and are hijacked by
viruses such as Ebola and HIV for their budding from
the cell
• MVB formation
• Cytokinesis
• Viral budding
Topologically similar
processes:
All away from the
cytosol (contrast to
budding of e.g. a
clathrin-coated vesicle
into the cytosol)
Molecular Biology of the Cell
Transfection methods to study
protein trafficking
Where is my protein of interest?
Tag with GFP
Transfect into cell
Where is it?
Protein trafficking pathways are saturable
Viruses exploit protein trafficking pathways
For example:
Some enveloped virus particles
are endocytosed and fuse
across endosome membranes
VSV: vesicular stomatitis virus
SFV: Semliki Forest virus
VEE: Venezuelan equine encephalitis virus
LCMV: Lymphocytic Choriomenengitis Virus
UUKV: Uukuniemi virus
EBOV: Ebola virus
White and Whittaker, Traffic 2016, 17(6):593-614
Protein translocation across membranes
• General principles: signal, receptor,
channel
• Specific examples: nuclear import/
export, mitochondrial import, ER
translocation
Summary Protein transport beyond the ER
• General principles: signals, vesicle
transport, membrane fusion
• Specific examples: ER retention,
secretion/ exocytosis, lysosomal targeting,
endocytosis
Finally…
Please take a look at the materials on Moodle
• Translocation exercise
• Crosswords
• Links
• Papers
Thank you listening!
