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Intracellular vesicular traffic I: The Secretory Pathway. Barth Grant Grant@biology.rutgers.edu 732-445-7339. Compartments enclose a space called the lumen within a lipid bilayer. Vesicular Transport Proceeds by Budding and Fusion. Kinetics of Secretion. Overview of the secretory pathway.
The Secretory Pathway
All secretory and endocytic compartments are topologically equivalent to the extracellular space.
Protein Folding Chaperones like BIP/Hsp70 Prevent Unfolded Proteins and Unassembled Multimers such as antibodies from exiting the ER prematurely.
Golgi to Endosome transport, PM to Endosome transport, and perhaps other transport steps.
COPII-coated vesicles leave the ER, uncoat and begin to fuse with one-another to form vesicular-tubular clusters.
These clusters associate with motor proteins that drag them along microtubules in an ATP dependent process.
Meanwhile retrograde transport removes certain components, purifying and concentrating the secretory cargo further.
Microtubule Tracks and Motor Proteins Guide Transport Vesicles.
Note the saltatory movement in straight lines.
Escaped ER-resident proteins are retrieved from the Golgi by KDEL receptors that recognize specific retrieval signals in ER proteins.
Lumenal proteins of the ER contain
Signals recognized by the KDEL receptor
(ERD2). E.g. HSC70, PDI
KDEL is necessary and sufficient.
The KDEL-receptor cycles between the ER and Golgi via COPII and COPI-coated vesicles.
KDEL-receptors bind to KDEL-bearing proteins in the low pH environment of the Golgi and release that Cargo in the neutral pH of the ER.
pH probably alters KDEL receptor conformation - regulating cargo binding and inclusion in COPI vesicles.
Note that specific modifications occur in specific subcompartments, because modifying enzyme localization is tightly controlled.
The state of protein modification can identify how far a protein has proceeded in transport.
M6P modification is the key to sorting certain lysosomal enzymes during biosynthesis.
GlcNAc phosphotransferase transfers N-acetylglucosamine phosphate to mannose on lysosomal
Enzyme (e.g. Cathepsin D).
A phosphodiesterase removes the GlcNAc, leaving the phosphorylated mannose.
Phosphorylated mannose is a signal recognized by the Mannose-6-phosphate receptor.
Sorting of lumenal proteins can occur by binding transmembrane receptors.
Lysosomal enzymes modified with M6P are bound by the lumenal domain of MP6R.
MP6R-lysosomal enzyme complexes are recruited into clathrin/AP1 coated pits.
Vesicles deliver the MP6R-lysosomal enzyme complexes to the late endosome.
MP6R recycles to the golgi.
Lysosomal enzymes are delivered to lysosomes.
Tradional Model - Golgi is a static organelle. Secretory proteins move forward in small vesicles. Golgi resident proteins stay where they are.
“Radical” Model - Golgi is a dynamic structure. It only exists as a steady-state representation of transport intermediates. Secreted molecules move ahead with a cisterna. Golgi resident proteins move backward to stay in the same relative position.
Temperature sensitive lethal mutations define this pathway.
Many novel proteins required for each of these steps were discovered in this genetic screen.
Mutant phenotype reveals earliest requirement, not necessarily every requirement.
Membrane and cargo molecules become concentrated in transport vesicles as they leave the ER.
Membrane proteins interact with components of the COPII coat through exit signals on their cytosolic tails.
Soluble proteins bind to certain concentrated membrane proteins known as cargo receptors.
Sar1 is a small GTPase that acts to recruit coat proteins.
Sar1-GDP is converted to Sar1-GTP by a transmembrane GEF.
GTP binding triggers insertion of fatty acid tail of Sar1 into membrane.
Sar1-GTP on the membrane recruits COPII proteins, resulting in membrane curvature.
Pinching-off occurs to release the free vesicle.
Golgi from WT (uninfected) + Golgi
from mutant cells (infected) + cytosol
+ ATP + 3H-N-acetylglucosamine. Measure 3H-modified viral protein as a measure of transport.
Fractionation of “Cytosol” allowed biochemical purification of budding and fusion activities:
COPI coat - budding, vesicle formation
Snare Complex - membrane fusion
NSF/Snap complex - snare recycling
50 nanometer vesicles
Vesicle SNARES (v-snares) and target membrane snares (t-snares) form cognate pairs that confer specificity on the fusion reaction.
V-SNARES are incorporated into transport vesicles during budding.
After fusion v-snares and t-snares remain tightly associated.
SNARE proteins mediate both specificity of fusion and the fusion process itself.
V-SNARE (vesicle), T-SNARE (target), and SNAP25 form a 4-helix bundle (coiled-coil).
Specific Rab proteins are required for specific fusion events. Their exact role is unknown, but they probably
function to recruit other effectors that participate in docking and fusion. Rab-GTP is the active form.
Neuronal snares of the synaptic plasma membrane have been particularly well characterized.
The conserved snare complex is composed of a four-helix bundle.
In this case one helix is contributed by the integral membrane v-snare synaptobrevin, one by the integral membrane t-snare syntaxin, and two by the peripheral membrane t-snare SNAP25.
NSF, a AAA-ATPase, associates with the snare complex via adaptor proteins and dissociates the complex in an ATP dependent manner.
Without this reaction snares can be used only once!
Virus binds to cell surface receptors modified with sialic acid.
The “fusion peptide” is buried within the HA protein at neutral pH. (Spring-Loaded)
The virus enters the endosomal pathway where the pH is lower.
At pH 5 HA protein undergoes radical conformational change, extending the hyrophobic
“fusion-peptide” into the target membrane, initiating fusion, releasing the viral DNA into the
cytoplasm. The V-SNARE/T-SNARE/SNAP25 “snare-pin” resembles the HA “hairpin” .
Binding of fusion peptide to HA2
Globular domains dissociate.
Loop segment forms a
Fusion peptide inserts into
Outer leaflet mixing.
Bilayer fusion - fusion pore
Migration/widening of pore
to complete fusion.
Snare-pairing drives water from between opposing membranes.
Lipids of the outer leaflets interact to form a stalk.
The formerly inner leaflets meet to form a bilayer. This is called hemifusion.
Rupture of the new bilayer creates the pores which expands to complete fusion.