Transcript Molecular Cell Biology 6/e

Lodish • Berk • Kaiser • Krieger • Scott • Bretscher •Ploegh • Matsudaira
MOLECULAR CELL BIOLOGY
SIXTH EDITION
CHAPTER 14
Vesicular Traffic, Secretion,
and Endocytosis
©
2008 W.
H.©Freeman
andand
Company
Copyright
2008
W. H. Freeman
Company
13.1 Techniques for studying the
secretory pathway
Genetic and Biochemical Approaches
1. Transport of a protein through
the secretory pathway can be
assayed in the living cells
a. G. Palade
pancreatic acinar cell: pause-chase and
autoradiography
5 min “hot” → “cold” for 1, 5, 10, 30, 60,
120 min
b. visualizing by fluorescence microscopy
a. G. Palade
pancreatic acinar cell: pause-chase and
autoradiography
5 min “hot” → “cold” for 1, 5, 10, 30, 60,
120 min
b. visualizing by fluorescence microscopy
a. Construct genes containing GFP + VSVG
(vesicular stomatitis virus glycoprotein,
mutant version) and transfect into cultured
mammalian cells
b. Synthesize VSVG on ER
c. At 40 Co, VSVG is misfolded and retained in
ER, at 32 Co, VSVG is folded correctly, and
transferred to Golgi and plasma membrane.
2. Detection of compartment- specific
oligosaccharide modifications
An another way to follow the transport of secretory
proteins: modifications of CHO side chains at different
stages of secretory pathway
3. Yeast mutants define major stages and many
components in vesicular transport
Many secretory vesicles stay in the space between membrane
and cell wall (invertase, sucrose → glucose and fructose).
Use temp sensitive mutant (sec) or double mutant to detect
the accumulation protein in ER or Golgi
4. Cell-free transport assays allow dissection
of individual steps in vesicular transport
proteins (enzymes) transport from one Golgi cisterna
to another
needs cytosolic extract, ATP, GTP, physiological
temperature
14.2 Molecular mechanisms of
vesicular traffic
1999 Nobel Prize Award: Gunter Blobel
donor organelle → target organelle
vesicle budding and fusion
Binding :
1. recruitment of a small GTP-binding
protein to a patch of donor membrane
coat protein
2. membrane cargo protein involving
membrane cargo-receptor protein
3. V-SNARE
4. soluble proteins
1. Assembly of a protein coat
drives vesicle formation and
selection of cargo molecules
COP II: RER → Golgi
COP I: (retrograde) Golgi-Golgi cisternae, cisGolgi → RER
Clathrin: Trans Golgi →late endosome
PM →late endosome
Trans-Golgi → plasma membrane ??
2. A conserved set of GTPase switch
proteins control assembly of different
vesicle coats
Small GTP-binding protein (GTPase) closed
related to assembly
COP I, Clathrin: ARF
COP II : Sar1
Model for the role of Sar1 in the
assembly and disassembly of COP II
coats
3. Targeting sequences on cargo proteins
make specific molecular contacts with
coat proteins
membrane sorting signals on membrane cargo
proteins
and different sorting signals for soluble cargo proteins
called luminal sorting signals
SNARE protein:
(Soluble NSF Attachment Receptor)
T-SNARE: Target SNARE,(syntaxin, SNAP-25)
V-SNARE: Vesicle SNARE (VAMP)
60 members in yeast and mammalian cells
R-SNARE: an arginine in the formation of SNARE
core (synaptobrevin)
Q-SNARE: a glutamine of SNARE core (syntaxin,
SNAP-25)
1. In lysosome study, fusion yeast and
eukaryotes have 20+ V-SNAREs and
T-SNAREs
2. Mutant study found that a particular
vesicle has its own SNAREs (special
target sequence)
14.3 Early stages of secretory
pathway
closer look at vesicular traffic
through ER and Golgi (antero-grade
and retrograde)
1. COPII vesicles mediate transport
from the ER to the Golgi
cell-free extracts of yeast RER incubated
with cytosol, ATP, nonhydrolyzed analog
of GTP → vesicles formed in ER→
proteins for coat polymerization
From mutation study, there are several
proteins involved in the polymerization
Cystic fibrosis : mutations of CFTR (ATP binding
cassette protein on the epithelium of lung, pancreas, sweat
glands etc.)
Contain a di-acidic sorting signal that binds to the Sec 24
subunit of the COP II coat for transport out of the ER
mutation on position on 508 can not bind to Sec 24, and
prevents normal transport of CFTR to the plasma
membrane and retained in ER
2. COPI vesicles mediate retrograde
transport within the Golgi and from the
Golgi to the ER
ER resident proteins (Bip, isomerase)
have KDEL (lys-asp-x-x) at C-terminal, if
escape from ER to Golgi, COPI can bring back
to ER
First discovered when isolated Golgi
incubated with solution containing ATP,
cytosol, and a nonhydrolyzing analog of
GTP.
Isolated vesicles and found they contain 7
polypeptide subunits (coatmers)
Temperature sensitive mutant to study the
functions of proteins
3. Anterograde transport through Golgi
occurs by cisternae progression
vesicles transport model → cisternal
progression model
enzymes from trans-Golgi to medialGolgi, and from medial- to cis-Golgi
EM studies
14.4 Later stages of the secretory
pathway
Vesicle-mediated trafficking from
Trans-Golgi network
1.
Vesicles coated with clathrin and/or adaptor
proteins mediate several transport steps
clathrin: fibrous clathrins inner layer: adaptor
protein complexes (AP)
Cyro-electron mocrographs of 1000 assembly
particles
↓
digital imaging
Cargo protein with tyr-x-x (hydrophobic aa) recruite
into clathrin/Ap1 and budding from trans Golgi
to plasma membrane
2. Dynamin is required for pinching off of
clathrin vesicles
using nonhydrolyzable derivative of GTP
or dynamin mutants
3. Mannose-6-phosphate residues target
soluble proteins to lysosomes
M-6-P is synthesized in the cis-Golgi (N-linked)
4. Study of lysosomal storage diseases
revealed key components of the lysosomal
sorting pathway
I-disease :
lack of N-acetylglucosamine phosphotransferase
(can’t form M-6-P) in lysosomes → glycolipids
become large inclusion bodies and accumulate in
the cell.
5. Protein aggregation in thr trans-Golgi may
function in sorting proteins to regulated
secretory vesicles
ACTH, insulin, trypsinogen aggregates
Chromogranin A, Chromogranin B,
Secretogranin II
6. Some proteins undergo proteolytic
processing after leaving the trans-Golgi
proproteins: soluble lysosomal enzymes,
membrane proteins (influenza hemagglutinin),
secretion protein (albunim, insulin glucagon,
yeast mating factor)
7. Several pathways sort membrane proteins to
the apical or basolateral region of polarized
cells
Polarized cells contain apical, basolateral, and
tight junctions
small intestine: absorption
stomach: acidification
fractionation and microscopic observations :
all these proteins from Trans-Golgi→ various
kinds of vesicles, different Rab, v-SNARE
→ targets
MDCK (Madin-Darby Canine Kidney)
14.5 Receptor-mediated endocytosis
and the sorting of internalized
proteins
1. phagocytosis: macrophage
2. invaginated vesicles :
a. pinocytosis
b. receptor-mediated endocytosis (LDL,
insulin, transferrin involving clathrin/AP2)
1. Receptors for LDL and other ligands contain
sorting signals that target them for
endocytosis
LDL receptor:
839 residues of glycoprotein ,
signal transmembrane segment,
short C-terminal (cytosol, sorting sequence)
longer N-terminal (extracellular) containing 7
cysteine-rich which bind to Apo B-100
Familial hypercholesterolemia:
LDL receptor (LDLR) → mutation (can’t form receptor)
→ LDLs internalization decreases and LDL
concentration in plasma increases
Homozygous: 6X plasma LDL as high as normal, die of
heart attack before late 20s
also mutation on C-terminal NPXY (Asn-Pro-X-Tyr)
sorting sequence → can not bind to AP2 complex →
clathrin coat can not form (no pits form)
other kinds of receptor mutations can not bind to LDL
2. The acid pH of late endosomes causes most
receptor-ligand complexes to dissociate
Model for pH-dependent binding of LDL particles
by the LDL receptor
3. The endocytic pathway delivers iron to cells
without dissociation of the receptor-transferrin
complex in endosomes
transport iron from the liver and from the intestine
to all the tissues of the body
14.6 Directing membrane proteins
and cytosolic materials to the
lysosome
Inward budding of the endosomal membrane
Cargo proteins destined to enter the multivesicular
endosome usually receive their ubiquitin tag
at the PM, the TGN, or the endosomal membrane
(cytosolic or misfolded ER proteins – ubiquitine to
proteosomes)
In the membrane of the endosome, a ubiquitintagged peripheral membrane protein, Hrs,
facilitates loading ubiquitin tag cargo protein into
vesicle buds directed into the interior of the
endosome.
1. Retroviruses bud from the plasma membrane
by a process similar to formation of
multivesicular endosomes
2. The autophagic pathway delivers
cytosolic proteins or entire organelles to
lysosomes
a. starvation
b. autophagy (eating oneself) double
membrane structure (from ER?) envelops an
entire organelle to form autophagosome
c. The autophagosome is organelle specific
(signal binding sites?)
d. Atg8 protein
e. targeting and fusion proteins (?)