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The Secretory Pathway
Becky Dutch
Molecular and Cellular Biochemistry
1. ER - translation
2. ER- protein modifications
3. Discussion Section
4. Golgi apparatus
5. Vesicular transport
Lecture 5:
Transport Vesicles
Reading: Alberts Chapter 13
Lodish Sections 17.10
Vesicular Transport
1. How are these vesicles
formed? How are
different proteins
incorporated?
2. How are these vesicles
targeted?
3. How do they fuse
with their target?
. Lodish, Fig 17-13
Three types of coated vesicles
known:
Each have different coat protein:
clathrin
COP I
COP II
Each involved in specific cellular
transport pathways
Lodish 17-50
Types of vesicles and their target locations
Lodish 17-51
All three types have similar vesicle budding:
Coat proteins polymerize around the cytosolic face of budding
vesicle
Coat and adapter proteins help select cargo
GTP-binding protein regulates the rate of vesicle formation
COP I vesicles
Coat protein formed from
coatamers: cytosolic complex
with seven subunits.
Polymerize on surface of vesicles
to drive formation. Dissociate
from vesicle after formation.
Golgi transport - retrograde
and likely anterograde
Also retrograde Golgi to ER
transport
Lodish 17-56
Cell-free system for studying Golgi transport
Cultured cells missing one of
the processing enzymes - this
will allow differentiation of the
two populations of Golgi
Lodish 17-57
Cell-free system for studying Golgi transport
Infect mutant cells with
VSV - makes only one viral
glycoprotein - VSV G
Addition of N-acetylglucos.
to VSV G wlll only happen
if transport to wt Golgi
stack occurs
Lodish 17-57
Assay used to identify and
study function of proteins
involved in Golgi vesicular
transport
Formation of COP I vesicles
Cell-free system just described
very helpful in determing roles
1. ARF - small GTPase, releases
GDP and binds GTP
- Golgi attached enyzme that
promotes this unknown
2. ARF-GTP binds receptors on
Golgi membrane
3. COP I coatamers bind to ARF,
other protein on cytosolic face.
4. Fatty acyl CoA helps budding
mechanism unknown.
5. If non-hydrolyzable GTP used vesicles form and release, but
Lodish 17-58 COP I never disassociates
Role of COP I vesicles
Retrograde transport - Golgi to ER
KDEL receptor and other membrane proteins to be returned to
ER - have KKXX sequence at end of C-terminus. This binds
COP a and b. This sequence necessary and sufficient to drive
transport to ER.
Yeast mutants lacking COP a and b can’t do retrograde transport
Retrograde transport - in Golgi
Moving specific proteins trans to medial, medial to cis
Anterograde transport in Golgi
COP I vesicles with lots of cargo, no KDEL - fast track
COP II vesicles
ER to Golgi transport
Cell-free extracts of yeast rough ER plus cytosol and ATP vesicles form - COP II
Formation - similar to COP I. Sec12 catalyzes exchange
of GDP for GTP in th Sar I protein. Complex forms with
Sec23 and Sec24 proteins, followed by binding of Sec13,
Sec31, then Sec16.
Contain a family of 24kDa proteins that selectively bind soluble
proteins bound for Golgi. Integral membrane proteins to
be transported generally have Asp-X-Glu sequence - binds to
one or more COP II proteins
Exocytosis: TGN to Cell Surface
Constituitive and Regulated secretion
Clathrin
vesicles
Alberts 13-36
Exocytosis of secretory vesicles
Secretory vesicles very densely packed - can release
large amounts of material
Regulated secretion - vesicles
move from TGN to site of
secretion. Can be a long
distance (nerve cells)
Triggered release - signal to
secrete can be hormone
binding receptor, electrical
excitation. Increases in
Ca2+ often important.
Alberts 13-39
Mast cell - example of regulated exocytosis
Histamine released in response to binding of specific ligands
Gives many of symptoms of allergic reactions
Mast cell
incubated
in solution
with ligand
-Response all
over cell
If ligand is
localized to
one spot response will
be localized.
Alberts 13-41
Targetting and Fusion
Common motifs for all types
of vesicles - fusion after
depolymerization; conserved
set of proteins that promote
targetting and fusion
V-SNARE - in transport vesicle important for targeting
T-SNARE - on target, along with
ubiquitous SNAP-25
V-SNARE, T-SNARE, SNAP25
form complex - fusion
Other proteins involved - NSF
(ATPase), SNAP proteins
Lodish 17-59
Rab proteins - regulators of vesicular traffic
Rab proteins - GTP binding proteins
Approx. 200 amino acids - structure similar to Ras
Bind and hydrolyze GTP - this is hypothesized to regulate
rate of vesicular fusion
GDI - catalyzes GDP/GTP exchange of Rabs - this leads
to conformational change in Rab that lets it bind vesicle
GTP hydrolysis leads to release of Rab after membrane fusion
Structures of many of these proteins recently
determined
Brunger, Curr. Op. Struct.
Biol. (2001) 11:163-173
Synaptobrevin=VAMP = v-SNARE; syntaxin=t-SNARE; synaptotagmin Ca2+ binding protein; SNARE complex = portions of synaptobrevin, syntaxin
and SNAP-25
SNARE complex has several states - zipper model
A - closed state; B - binary - syntaxin, SNAP25; C; D - ternary with synaptobrevin in complex
Viral fusion proteins - best-understood examples
Single protein systems which promote high level membrane fusion
Syncytia assay of wt SV5 F and the F Tail- mutant.
Viral fusion proteins undergo conformational
changes upon triggering of fusion
Lodish 17-60
Similar complexes containing heptad repeat
regions form in a number of viral fusion proteins
Formation of these heptad repeat complexes
critical for membrane fusion
Steps for fusion pore formation
A group of pH activated HA spikes work
together to form fused membranes
HA protein inactive after this process unlike SNARES, which recycle
Lodish 17-61
Relation of SNARE to viral fusion proteins
Complexes containing
coiled-coils fundamental
to both systems
Skehal and Wiley, Cell (1998) 95: 871-874
Secretory pathway critical for cellular
function
. Lodish, Fig 17-13