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6. Protein targeting and membrane
traffic








Principles of intracellular organization
Signal sequences and targeting signals
Membrane translocation mechanisms
Tubulo-vesicular membrane traffic
Molecular mechanisms of vesicle budding
Vesicle transport along microtubules
Molecular mechanisms of membrane fusion
Neurotransmitter release at neuron synapses
1
(Eukaryote) cells are complex
10 µm
Dual-Core Intel®
Xeon® processor
Bovine pulmonary endothelial
cell
• 37 x 37 mm
• 8.2 108 transistors
• 45 nm manufacturing
technology
• 3 GHz clock speed
• 50 x 50 x 5 µm
• about 104 different proteins
(out of 106 possible)
• average protein size 5 nm
• typical protein turnover 100 Hz
Fast execution of
consecutive
instructions
Able to duplicate
itself and to adapt to
its environment
2
Microprocessor vs (eukaryote) cells
Dual-Core Intel®
Xeon® processor
Bovine pulmonary endothelial
cell
• 37 x 37 mm
• 8.2 108 transistors
• 45 nm manufacturing
technology
• 3 GHz clock speed
• 50 x 50 x 5 µm
• about 104 different proteins
(out of 106 possible)
• average protein size 5 nm
• typical protein turnover 100 Hz
Communication
10-10 s
diffusion limited
(DGFP = 90 µm2.s-1  27 s
Light speed
Hink et al. JBC 275 :17556–17560)
Number of components
8.2 108
about 1010 proteins
1 pM < concentration < 100 µM (actin)
1-108 molecules/cell
How is cell synchronization achieved ?
How do proteins localize properly ?
3
Principles of cellular organization
 Structural elements : plasma membrane, nucleus, organelles, vesicles,
cytoskeletons, ciliae
 spatial organization of the cell
- The range of interaction between macromolecules inside the cell is very short,
typically a few nanometers.
- Macromolecular complexes (membranes, polymers, complexes) are needed to
organize the cell’s interior.
- The dynamics of macromolecular complexes is coupled to energy
consumption and is able to exert forces and flows sufficient to shape the cell
- Proteins are targeted to specific structural elements thanks to targeting signals
 Roles of the different structural elements : energy production, macromolecule
synthesis and degradation, intracellular transport, uptake, secretion, movement,
cell division
 Functional organization of the cell
- Membranes delineate cell compartments where different reactions take place.
- Membranes and polymers accelerate reactions by reducing the dimensionality
of the space where diffusion takes place
4
Localization of main cellular functions
plasma membrane
transport
cell adhesion (toward the
ECM and other cells)
endocytosis
nucleus
import and
export
DNA
replication
and repair
RNA
synthesis
exocytosis
lipid -oxidation
citric acid cycle
oxidative
phosphorylation
mitochondria
vesicular
transport
lipid
synthesis
glycolysis
protein
synthesis
5
Why intracellular traffic is important ?
 Diffusion is effective at nm-µm distances  self-assembly
 Above 10 µm, diffusion is very ineffective  energy driven assembly and
movement
Diffusion kinetics : r2(t) = Dt
Einstein-Schmoluchowski
D = kBT/6phR  M-1/3
for globular proteins
D (µm2.s-1)
Water
3000
+
Na
1000
Acetylcholine
200
Insulin (5,7 kDa)
150
GFP (30 kDa)
90
Myosin (250 kDa)
10
r@1msec (µm)
1.7
1
0.5
0.4
0.24
0.027
r@1sec (µm)
55
32
14
12
9
1
 Long range organization within the cell : polymers, membrane curvature,
mechanical forces  self-organization of intracellular compartments
 Long range organization within tissues : fluid convection, fluid flow,
mechanical forces
6
Protein targeting and sorting signals
 Targeting and sorting signals are amino acid stretches encoded in
the primary sequence that define the journey of a given protein in the
cell and its final localization. A single protein may contain several
targeting and sorting signals.
A signal sequence consists of about 20 amino acids at the N-terminal
end of the primary sequence of a protein. It allows insertion of the
protein in the membrane of an organelle (endoplasmic reticulum,
mitochondria...) or translocation of the protein through one or several
organelle membranes. When the protein is imported inside the lumen of
the organelle, the signal sequence is often cleaved by a specific
protease and degraded.
 Retention signals maintain some proteins in a given compartment,
usually by interacting with specific membrane receptors. The steadystate localization of these receptors results from the balance of
anterograde and retrograde traffic.
 Targeting signals are either constitutive (always active) or may be
activated by phosphorylation and/or conformational changes.
7
Example of targeting signals
Target compartement
import in the ER
Typical sequence
+MMSFVSLLLVGILF WATEAEQLTKCEVFN
retention in the ER
Import in mitochondria
KDEL+MLELRNSIRFFKPATRTLCSSRYLL
Import in the nucleus
PPKKKRKV
Import in peroxysomes
SKL
Sorting to endosomes
ou lysosomes
YxxP, ExxLL
plasma membrane
Acylation (modification by a lipid group)
+GSSKSKPK
CxxL-, CCxx-
plasma membrane
membrane proteins and
secreted proteins
glycosylation at NxS/T sequence
Hydrophobic amino acid
Negatively charged amino acid
Positively charge amino acid
8
Targeting signal characteristics
 They are specific of a given compartment and well conserved in eukaryotes
 They allow a reversible interaction with the proteins organizing the intracellular
protein transport
 They are generic and allow targeting of a protein of interest
 In the absence of any signal, a protein may be targeted to a default localization
Example : human cytochrome c oxydase subunit VIII (COX-8)
cleavage
10
20
30
40
50
60
MSVLTPLLLR GLTGSARRLP VPRAKIHSLP PEGKLGIMEL AVGLTSCFVT FLLPAGWILS
69
HLETYRRPE
Key
From
TRANSIT
1
CHAIN
26
TOPO_DOM
26
TRANSMEM
37
TOPO_DOM
61
To Length
25
25
69 44
36 11
60 24
69 9
Description
Mitochondrion signal sequence
Cytochrome c oxidase polypeptide VIII-liver/heart.
Mitochondrial matrix
transmembrane domain
Mitochondrial intermembrane
9
Example of a plasmid used to
label mitochondria
‘Living Colors’: set of
plasmids encoding fluorescent proteins
targeted to specific subcellular
compartments
Cytomegalovirus promoter (PCMV)
Coding sequence :
COX-8 sequence signal
Ds-Red2 coding sequence
MSVLTPLLLR GLTGSARRLP VPRAKRSSKN VIKEFMRFKV RMEGTVNGHE FEIEGEGEGR PYEGHNTVKL
KVTKGGPLPF AWDILSPQFQ YGSKVYVKHP ADIPDYKKLS FPEGFKWERV MNFEDGGVVT VTQDSSLQDG
CFIYKVKFIG VNFPSDGPVM QKKTMGWEAS TERLYPRDGV LKGEIHKALK LKDGGHYLVE FKSIYMAKKP
VQLPGYYYVD SKLDITSHNE DYTIVEQYER TEGRHHLFL
Terminator sequence of SV40 virus
(SV40polyA)
10
Protein targeting and membrane
traffic








Principles of intracellular organization
Signal sequences and targeting signals
Membrane translocation mechanisms
Tubulo-vesicular membrane traffic
Molecular mechanisms of vesicle budding
Vesicle transport along microtubules
Molecular mechanisms of membrane fusion
Neurotransmitter release at neuron synapses
11
Mechanisms of membrane translocation
Example : mitochondrial import
Experimental evidence using semi-reconstituted systems
proteolysis and SDS-PAGE analysis
reveal the existence of a translocation
intermediate
12
Protein import pathways into mitochondria
Wiedemann, N. et al. J. Biol. Chem. 2004;279:14473-14476
13
Protein translocation into the endoplasmic reticulum
ribosome
messenger
RNA
GDP
GTP
Signal
sequence
Signal Recognition
Particle and
ribosomal receptor
translocator
specific
peptidase
mature polypeptide
for secretion 14
Soluble protein secretion
GE Palade and JD Jamieson experiment 1969
pulse : 1 min
[3H]-leucine
3 min chase
20 min chase
90 min chase
Observation of
newly
synthesized
proteins by
autoradiography
and electronic
microscopy
endoplasmic
reticulum
Golgi apparatus
secretion
vesicles
15
Plasma membrane
recycling
endosome
late
endosome
secretory
vesicles
lysosome
Retrograde traffic
TGN
trans-Golgi
medial-Golgi
cis-Golgi
exocytosis (secretion)
endocytosis
sorting endosome
EGTC
endoplasmic reticulum
Nucleus
16
Glycosylation of secreted proteins and of transmembrane
proteins targeted to the plasma membrane
 Secreted soluble proteins and transmembrane proteins targeted to the plasma
membrane are post-translationally modified by sugar groups (glycosylation).
 Glycosylation consists of a complex oligosaccharide linked to an asparagine in the
sequence NxS/T.
 Glycosylation sequentially takes place in the endoplasmic reticulum and in Golgi
apparatus cisternae.
17
Protein modification in the Golgi apparatus
Oligosaccharide
attached to an
asparagine amino
acid
Intra-Golgi compartments
Ultrastructure of the Golgi apparatus 18
Constitutive and regulated secretion
Yeast enzyme
secretion
Synaptic
neurotransmitter
secretion
Insulin secretion
19
Molecular study of membrane intracellular traffic
Identification of proteins involved in membrane traffic :
1. Genetic approach : screening of thermosensitive secretion
mutants in Saccharomyces cerevisiae
2. Biochemical approach : in vitro reconstitution of cis-medial
intra Golgi transport and purification of soluble factors
Study of protein interactions :
Interaction between protein partners : co-immunoprecipitation
Intracellular localization of proteins : optical microscopy
(immunolocalization, FRET) and (immuno) electron microscopy
In vitro reconstitution of membrane traffic :
In vitro reconstitution of membrane fusion and membrane budding
using synthetic vesicles and recombinant proteins
In vitro reconstitution of vesicle-cytoskeleton interaction and
directed transport
Study of regulations :
Intracellular specificity of membrane traffic
Specific toxins inhibit membrane traffic
20
1. Screening for thermosensitive secretion mutants generated
by random mutagenesis in the yeast Saccharomyces cerevisiae
R. Scheckman, P. Novick
Random generation of
mutant cells
Thermosensitive
mutants
23 °C
Permissive
temperature
36 °C
Restrictive
temperature
Selection
screen
enzyme secretion at
the restrictive
temperature
sec mutants (contain a thermosensitive
21
version of a Secx protein)
Complementing the molecular defect of thermosensitive mutants
secx thermosensitive mutant
generated in a TRPD strain
constitutive
promoter
transformation
with an
expression
library
random
cDNA
TRP
Selection at the
restrictive
temperature in
the absence of
tryptophan
TRPD : yeast strain lacking a
TRP gene used for
tryptophane biosynthesis,
which is unable to grow in the
absence of tryptophan
(auxotroph strain)
Most frequent cDNA :
wild type gene secx
Less frequent cDNA :
suppressor genes secy
cDNA analysis
22
Sec mutant morphology
Sec mutants are classified in five classes according to yeast ultrastructure observed by
electron microscopy
transport vesicles 
plasma membrane
endoplasmic reticulum 
Golgi apparatus
Golgi apparatus 
transport vesicles
23
2. Using in vitro reconstituted cell free systems to study
vesicular traffic
J.E. Rothman
 principle
 identification of proteins involved in intra-Golgi membrane traffic
Identification of the targets of biochemical inhibitors
Cytosol fractionation and reconstitution
GTPgS→ARF, coatomer
NEM→NSF, SNAP, SNAREs
24
 experimental design
CHO-15B cells (deficient
in N-acetyl glucosamine VSV-G protein transport
transferase) infected by intermediates contained
the vesicular somatitis in the cis cisternae of the
virus (VSV)
Golgi stack
Wild type CHO cells
(contain active N-acetyl
glucosamine transferase)
Purification
of Golgi
stacks
Cytosol
preparation
Golgi : donor fraction
Golgi : acceptor fraction
Cytosol, ATP, UTP
UDP-[3H] N-acetyl glucosamine
Incubation
37°C
Detergent lysis +
Immunoprecipitation
with anti-VSV-G
antibodies
Quantitation of [3H] Nacetyl glucosamine
associated to VSV-G
25
Biochemical study of protein interaction : co(immuno)précipitation
or pull-down techniques
1. Cell extract
(membranes
solubilized with a
detergent)
2. Affinity
chromatography
Antibodies are
often used !!!!
Purification with
immobilized antibodies
Purification of protein
complexes
Use of magnetic beads
reduces contamination
with aggregated
proteins
3. Identification of the eluted
polypeptides by Western blot or
mass spectrometry
26
Protein targeting and membrane
traffic








Principles of intracellular organization
Signal sequences and targeting signals
Membrane translocation mechanisms
Tubulo-vesicular membrane traffic
Molecular mechanisms of vesicle budding
Vesicle transport along microtubules
Molecular mechanisms of membrane fusion
Neurotransmitter release at neuron synapses
27
1. Vesicle budding
GTP
hydrolysis
(ARF)
Bonifacino et Glick 2004 Cell 116: 153-166
28
Inhibition by the GTPgS or by an
ARF mutant unable to hydrolyze
its bound GTP
Dt
120 min - Dt
+ GTPgS
standard assay
Tanigawa et al. 1993
J Cell Biol. 123 : 1365-1371
standard assay
+ GTPgS
29
Accumulation of coated vesicles in the presence of GTPgS
yeast
orthologue
Sec33
Sec26/27
Sec21
Sec28
Sar1p
ARF
coatomer
COPI coat
 active in the presence of GTPgS
J.E. Rothman & L. Orci
30
Assembly and disassembly of protein coat is coupled to
GTP hydrolysis by ARF, a small G protein
Example : COP1 (coatomer)
Cargo : proteins to be transported
Coatomer : a protein complex that
binds to the membrane, selects
the cargo and deforms the
membrane
GTPgS
ARF (ADP ribosylating factor) : a
small G protein
GEF : G protein exchange factor
Three types of coat exist : clathrin (endocytosis), COPI (secretion, retrograde traffic) and COPII (secretion,
anterograde traffic)
31
Model for Membrane Recruitment of Coatomer
(A)
a composite model of βδ/γζ-COP bound to membrane
via two molecules of Arf1-GTP. The γζ-COP/Arf1
crystal structure is colored as is the second molecule
of Arf1. The remainder of βδ/γζ-COP, in grey. The Nterminal amphipathic α helices of Arf1 (colored red)
are modeled in their expected locations as membrane
anchors.
(B) Effects of mutations in the Arf1-binding sites of full-length
βδ/γζ-COP complex, measured using the pull-down
assay. Single mutations reduce but do not abolish
Arf1 interaction (lanes 3 and 4), whereas a double
mutation binds to Arf1 at background levels (compare
lane 5 to lane 1).
(C) The effects of single and double mutations in the βδ/γζCOP complex on GTP hydrolysis in the fluorescence
assay.
3-O-[N-methyl-anthraniloyl]-GTP
+ Arf-GAP
 mant-GTP fluorescence level
 mant-GDP fluorescence level
Xinchao Yu, Marianna Breitman, and Jonathan Goldberg. A Structure-Based Mechanism for Arf1-Dependent Recruitment of Coatomer to Membranes
Cell. 2012 148: 530–542.
Partial Model of Coatomer structure
A
(A)
Surface representation of the αβ’-COP triskelion. α-COP and β’-COP
subunits are colored in three shades of orange and green, respectively.
(B)
Known and unknown
elements of the αβ’εCOP complex. The
speculative element of
the diagram is the
dimer contact
(indicated by the
question mark) that
brings together two
copies of αβ’ε-COP.
(C) and (D) Model of an
icosahedral COPI
cage. This type of
structure is formed by
clathrin in vitro
C
Changwook Lee and Jonathan Goldberg. Structure of Coatomer Cage Proteins and the Relationship among COPI, COPII and Clathrin Vesicle Coats
Cell. 2010 142 : 123–132.
Guanosine-5’(g-thio)triphosphate or GTPgS is a nonhydrolyzable GTP analogue
S
 Other non hydrolyzable analogues exist : GppNHp
 GDPS is a GDP analogue that cannot be phosphorylated by endogenous
nucleoside diphosphate kinase (NDPK) :
ATP + GDP → ADP + GTP
34
Arf-GDP structure
1rrf
Gln71
Asp67
 phosphate
Mg2+
a phosphate
5Å
GDP
35
Small G protein signalling
 G proteins have two states (GDP or GTP bound)
 GEF and GAP controls the transition frequency over two orders of magnitude
 Specific amino acids (e.g. ArfQ71) are involved in GTP binding or hydrolysis
SIGNAL IN
GDP/GTP
+
exchange
G protein Exchange
Factor (GEF)
SIGNAL IN
-
GTP
hydrolysis
GTPase activating
protein (GAP)
36
Function
Vesicle transport and fusion
Cell signaling
Vesicle formation
Actin cytoskeleton regulation
Nucleocytoplasmic transport of
RNA and proteins
Wennerberg K et al. J Cell Sci 2005;118:843-846
2. Vesicle transport along microtubules
ATP/GTP hydrolysis (molecular motors)
4’ directed transport
Bonifacino et Glick 2004 Cell 116: 153-166
38
The tubulin cytoskeleton
Microtubules are tubulin polymers, a protein that binds and hydrolyzes GTP.
The cycle of microtubule polymerisation-depolymerisation is coupled to GTP
hydrolysis
Microtubules are organized radially around the centrosome, also called the
MicroTubule Organizing Center (MTOC) which is localized near the nucleus.
They allow centripetal or centrifuge organelle transport within the cell. During
cell division, the centrosome replicates and organizes chromosome separation
by forming a mitotic spindle.
Microtubules are rigid and their polymerization exert mechanical forces that
allow the centrosome to reach a position determined by the microtubule
depolymerization activity along the cell cortex (cell polarization).
Untreated cell
cellule treated with nocodazole, a
microtubule polymerization inhibitor
39
Tubulin and microtubule structure
5 nm
Tubulin is incorporated in protofilaments as TaGTPTGTP
The tubulin  subunit rapidly hydrolyses GTP into GDP
TaGTPTGDP protofilaments are instable
40
Microtubule instability
CENTROSOME
Microtubules minus ends are locked at the centrosome.
Microtubule plus end depolymerizes about 100 times faster when it contains GDP
tubulin than when it contains GTP tubulin. A GTP cap therefore, favors growth.
When it is lost, fast depolymerization occurs.
Individual microtubules therefore alternate between a period of slow growth and
a period of rapid disassembly, a phenomenon called dynamic instability.
- end
+ end
41
Microtubule dynamics
42
Molecular motors
 Kinesins are
molecular motors
that move to the +
end of microtubules
(anterograde)
 Dyneins are
molecular motors
that move to the end of microtubules
(retrograde)
Further readings :
http://www.ncbi.nlm.nih.
gov/books/NBK21710/
43
Höök P , Vallee R B J Cell Sci 2006;119:4369-4371
©2006 by The Company of Biologists Ltd
3. Membrane fusion
GTP
hydrolysis
(Rab)
ATP hydrolysis (NSF)
Bonifacino et Glick 2004 Cell 116: 153-166
45
Identification of a protein complex involved in membrane fusion
Inhibition of intra Golgi transport by N-ethyl maleimide, a molecule reacting with SH groups
 purification of NSF (NEM-Sensitive Factor), a soluble ATPase = Sec18
Purification of soluble proteins necessary for NSF binding to membranes
 identification of SNAP (Soluble NSF Attachment Proteins) = Sec17
Purification of transmembrane proteins necessary for NSF binding to membranes, from
brain membranes
 identification of SNAREs (Soluble NSF Attachment Proteins REceptors)
 Syntaxin
 SNAP-25 (25 kDa Synaptosomal Associated Protein)
 VAMP (vesicle-associated membrane protein)
46
N-ethylmaleimide
a-SNAP Soluble NSF Attachment Protein
N-ethylmaleimide Sensitive Factor (NSF)
Co-immunoprecipitation of a NSF-SNAP-SNAREs complex
Söllner et al. 1993 Nature 362: 319-324
syntaxin B
g-SNAP
syntaxin A
a-SNAP
SNAP-25
2
Myc-epitope: EQKLISEEDL
3
VAMP/
Synaptobrevin2
SDS-PAGE + mass spectrometry
48
Sutton et al. 1998 Nature 395: 347-353
The SNARE model
 SNAREs are present on transport
vesicles (v-SNAREs) and on target
membranes (t-SNARE).
 The specificity of membrane fusion
in ensured by the formation of specific
v-t SNARE complexes.
 SNAREs form a coiled-coil structure
consisting of 4 a helices that catalyses
membrane fusion (fusion complex).
 After membrane fusion, NSF and
SNAP proteins catalyze SNARE
disassembly.
 The cycle of SNARE complex
assembly and assembly is driven by
ATP hydrolysis.
49
In vitro reconstitution of membrane fusion
Donor
NBD-PE +
Rhodamine-PE
(2%) 50 nm
v-SNARE
750/vesicle
Acceptor
PC, PS, PE
15 x excess
t-SNARE
75/vesicle
+Triton X-100
Weber et al. 1998 Cell 92 : 759-772
50
Retrograde traffic
exocytosis (secretion)
51
endocytosis
Protein targeting and membrane
traffic








Principles of intracellular organization
Signal sequences and targeting signals
Membrane translocation mechanisms
Tubulo-vesicular membrane traffic
Molecular mechanisms of vesicle budding
Vesicle transport along microtubules
Molecular mechanisms of membrane fusion
Neurotransmitter release at neuron synapses
52
Vesicular transport summary : energy consumption
ATP/GTP hydrolysis (molecular motors)
4’ directed transport
GTP
hydrolysis
(ARF)
GTP
hydrolysis
(Rab)
ATP hydrolysis (NSF)
Bonifacino et Glick 2004 Cell 116: 153-166
53
At the synapse, neurotransmitters are released by controlled
membrane fusion
0.5 msec
54
 Pre-assembled SNARE complexes held
by synaptotagmin
 Voltage driven Ca2+ entry
 Synaptotagmin activation by Ca2+
 SNARE-driven membrane fusion
55
Specific neurotoxins cleave neuronal SNARE proteins
Syntaxin
VAMP
SNAP-25
TeNT, tetanus neurotoxin
BoNT, botulism neurotoxin
G Schiavo (2000 ) Physiological Reviews, 80: 717-766
56
The Botox
The BoNT/A toxin blocks the synaptic
transmission between nerves and
muscles. Intra-muscular injection
relaxes muscles of the face.
Hyaluronic acid is a natural polymer
of the extracellular matrix. Crosslinked hyaluronic acid is
intradermically injected to smooth the
face surface
The effect lasts 3 to 4 months; a single injection costs about 400 €.
Botox is often combined with a product to fill wrinkles.
57
G Palade
NP 1974
R Scheckman
C de Duve
L Orci
A Claude
G Blobel
NP 1999
J Rothmann
58
i-bio Seminars
Wittinghofer : small G proteins
Scheckman : membrane traffic
Model for Membrane Recruitment of Coatomer
(A)
a composite model of βδ/γζ-COP bound to membrane
via two molecules of Arf1-GTP. The γζ-COP/Arf1
crystal structure is colored as is the second molecule
of Arf1. The remainder of βδ/γζ-COP, in grey. The Nterminal amphipathic α helices of Arf1 (colored red)
are modeled in their expected locations as membrane
anchors.
(B) Effects of mutations in the Arf1-binding sites of full-length
βδ/γζ-COP complex, measured using the pull-down
assay. Single mutations reduce but do not abolish
Arf1 interaction (lanes 3 and 4), whereas a double
mutation binds to Arf1 at background levels (compare
lane 5 to lane 1).
(C) The effects of single and double mutations in the βδ/γζCOP complex on GTP hydrolysis in the fluorescence
assay.
3-O-[N-methyl-anthraniloyl]-GTP
+ Arf-GAP
 mant-GTP fluorescence level
 mant-GDP fluorescence level
Xinchao Yu, Marianna Breitman, and Jonathan Goldberg. A Structure-Based Mechanism for Arf1-Dependent Recruitment of Coatomer to Membranes
Cell. 2012 148: 530–542.
Partial Model of Coatomer structure
A
(A)
Surface representation of the αβ’-COP triskelion. α-COP and β’-COP
subunits are colored in three shades of orange and green, respectively.
(B)
Known and unknown
elements of the αβ’εCOP complex. The
speculative element of
the diagram is the
dimer contact
(indicated by the
question mark) that
brings together two
copies of αβ’ε-COP.
(C) and (D) Model of an
icosahedral COPI
cage. This type of
structure is formed by
clathrin in vitro
C
Changwook Lee and Jonathan Goldberg. Structure of Coatomer Cage Proteins and the Relationship among COPI, COPII and Clathrin Vesicle Coats
Cell. 2010 142 : 123–132.
Function
Vesicle transport and fusion
Cell signaling
Vesicle formation
Actin cytoskeleton regulation
Nucleocytoplasmic transport of
RNA and proteins
Wennerberg K et al. J Cell Sci 2005;118:843-846
N-ethylmaleimide
a-SNAP Soluble NSF Attachment Protein
N-ethylmaleimide Sensitive Factor (NSF)