vesicle formation and targeting is a multi-step process 3. targeting and docking to specific...
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Vesicle formation and targeting is a multi-step process
3. Targeting and docking to specific compartment…
2. Formation of coated transport vesicle…
GAP and Sar1
Donor compartment
1. Formation of coated buds…
(ATP, GTP, and cytoplasmic protein factors…)
GEF and Sar1
Target compartment
4. Uncoating…
GTP
GDP + Pi
Coat proteins (“COPs” or
“coatomer”)
SNAREs and Rabs
5. Fusion…SNARE plus other fusion
proteins
Lecture 16
Vesicle transport and targeting in the secretory pathwayCOP coated vesiclesSNAREs
Protein sorting/targetingSecretion - Golgi to plasma membraneRetention in ERGolgi to lysosome
How are proteins sorted to appropriate vesicles so that they are transported to proper location?
What are the address labels?
Two secretory pathways; constitutive and regulated
Default pathway for ER/Golgi proteins
If no address label, then secrete
Signal required to trigger secretory granule fusionExample - neurotransmitter release
ECB 15-28
Inside lumen is equivalent to outside of cell
Regulated secretion
Secretory granules containing insulin in pancreatic cells Signal for release is elevated glucose levels in blood
If secretion is default, how are resident ER proteins retained?
C, M, T Golgi
ER
Plasma membrane
OutsideCGN TGN
Constituitive secretion
Secretory granuleRegulated secretion
Ex: BiP is a member of the HSP70 family that functions in the ERThey
aren’t!
BiP escapes from ER and must be “retrieved” from the Golgi…
C-terminal KDEL in BiP sequence functions as retrieval signal…
BiP KDEL
KKXX
KDEL-R
KDEL-receptors in Golgi direct retrieval/recycling…
KKXX at C-terminus of KDEL-R binds COPI coat and targets back to ER…
Summary so far of protein targeting, revisited…
Cytoplasm
Secretion/membrane proteins
Secretory vesicles
RetrievalTransport
(constituitive secretion)
(regulated secretion)
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RER
Golgi
Plasma membrane
See ECB figure 14-5
Default
Signal sequence (hydrophobic -helix)
KDEL (soluble proteins)
KKXX (membrane proteins)
Lysosomes
?Default
How are proteins targeted to the lysosome?
Lecture 16
Vesicle transport and targeting in the secretory pathwayCOP coated vesiclesSNAREs
Protein sorting Secretion - Golgi to plasma membraneRetention in ERGolgi to lysosome
How are proteins sorted to vesicles leaving TGN for lysosome?
Lysosomes degrade and recycle macromolecules…
Lysosomes in plant and animal cells contain acid hydrolases (hydrolytic enzymes) for degrading/recycling macromolecules
pH of lumen is about 5 - acidic!
How are hydrolases and other proteins targeted to lysosomes?
ECB 15-34
I-cell disease helped decipher the signal for targeting proteins to the lysosome
• Recessive mutation in single gene…
• Fibroblasts of patients contain large inclusions (I-cells)…
• Lysosomes lack normal complement of acid hydrolases…
• All lysosomal enzymes secreted (secretion is the “default” fate for proteins in the ER-Golgi pathway)…
• Lysosomal enzymes of “wild-type” (normal) cells are modified by phosphorylation of mannose on oligosaccharide (forming mannose-6-phosphate)…
• Lysosomal proteins of I-cells lack M-6-P…
• Lysosomal targeting signal resides in carbohydrate!
Mannose-6-P targets proteins from Golgi to lysosomeCis Golgi
Network (CGN)Trans Golgi
Network (TGN)
RER
M6P receptor recycling back to Golgi
Transport via clathrin-coated vesicles to…
Lysosome
M6P receptor in TGN directs transport of enzymes to lysosome via clathrin-coated vesicles
Addition of M6P to lysosomal enzymes in cis-Golgi
Patients with I-cell disease lack phosphotransferase needed for addition of M-6-P to lysosomal proteins in fibroblasts… secreted…
Lysosomal hydrolase (precursor
)
Addition of M6P
Removal of phosphate &proteolytic processing…
Maturehydrolase
M6P receptor
Clathrin coat
Uncoupling(pH 5)
Protein targeting, revisited
Next lecture: endocytosis and clathrin coats
Cytoplasm
Secretion/membrane proteins
Secretory vesicles
Lysosomes
RetrievalTransport
(constituitive secretion)
(regulated secretion)
Pro
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Vesi
cle t
arg
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RER
Golgi
Plasma membrane
See ECB figure 14-5
Signal sequence (hydrophobic -helix)
KDEL (soluble proteins)
KKXX (membrane proteins)
M6P
Default or
signal?
Default or
signal?
Lecture 17
The pathways to the lysosomePhagocytosisAutophagyEndocytosis
Endocytosis- The inward limb of membrane cyclingPinocytosis
Clathrin coated vesiclesReceptor-mediated endocytosis
Three pathways to the lysosome
PhagocytosisEndocytosisAutophagy
ECB 15-35
Bacterium
Phagocytosis - “cell eating”
Phagosome
Lysosome
1. “Phagocytosis”
A. Entrapment by pseudopods
B. Engulfment: pseudopods fuse to internalize prey in phagosome…
C. Digestion: phagosome fuses with lysosome
Pseudopods
Vesicles wlysosomal enzymes
Some bacteria have evolved to evade digestion in lysosomes, and live as intracellular parasites or pathogens…
Myxobacteria tuberculosis (tuberculosis)…
Listeria monocytogenes (listeria)…
Yersinia pestis (plague)…
Performed by specialized “phagocytes:”
WBCs
Where do vesicles with lysosomal contents come from? What is their address label?
Autophagy (“Self-eating”); used to recycle worn-out organelles
Phagosome
Worn outmitochondrion
1. “Phagocytosis”
2. “Autophagy”Autophagosome
Bacterium
LysosomeWorn out organelle engulfed by EREndoplasmicreticulum
Vesicles wlysosomal enzymes
Endocytosis:
Phagosome
Lateendosome
Earlyendosome
Endoplasmicreticulum
1. “Phagocytosis”
3. “Endocytosis”
2. “Autophagy”Autophagosome
Bacterium
Worn outmitochondrion
Vesicles wlysosomal enzymes
Endocytotic vesicles
Lysosome
Pinocytosis (“cell drinking”) and “receptor-mediated” endocytosis
Note that vesicles from TGN targeted to lysosome by M6P actually fuse with precursor vesicles/organelles to form lysosome
Overview of “pinocytosis” (“bulk” or “fluid-phase” endocytosis)
ATP
ADP+Pi
H+
ATP
ADP+Pi
H+
ATP
ADP+Pi
H+
Fluid-phase endocytosis can be followed in live cells with fluorescent dyes
~30’: Lysosome(pH<5)…
~ 5’: late endosome(pH 5.5~6)…
~ 1’: early endosome(pH~6)…
Coated pit
ATP ADP+Pi
Uncoating(seconds)…
GTP
GDP+Pi
Coatedvesicle
Proteinaceous coat As many as ~2500 coated
vesicles/min (~2-3% of surface area)!
Delivery of acid hydrolases from
TGN…Early endosome - late endosome - lysosome is a continuum
EM views - coated pit to coated vesicle
Coated pits coated vesicles
ECB 15-18
Protein coat is “geodesic” clathrin cage
3 clathrin “heavy chains”
(~180-190 kDa)…
…plus…
3 clathrin light chains(~40 kDa)…
…form…
“Triskelions”…
Spontaneously assemble into “geodesic” vesicle coats…
Clathrin “heavy chain”
“Light chain”
15.8-clathrin.mov
ECB 15-19
clathrin
adaptins
membrane
receptors
“cargo”
Components of a clathrin-coated vesicle
Cargo and receptors we know from COP-coated vesicles
Adaptins - adaptors that bind clathrin and cargo receptor, thereby regulating which cargo gets loaded into clathrin-coated vesicle
ECB 15-19
Coated pit “pinchingoff”
(dynamin)
budding uncoating
Pinching off of vesicles requires the protein dynamin
Assembly of coat causes pit to form due to 3D shape of clathrin coat
ECB 15-19
dynamin
GTP
GDPGTPase that regulates pinching off
Dynamin is a GTPase
Explains why non-hydrolyzable GTP analogues block endocytosis
GTP
GDP + Pi
Clathrin uncoating ATPase
Naked transport
vesicle
Dynamin
ATP
ADP + Pi
Clathrin-coated vesicles are rapidly uncoated
By the “clathrin-uncoating ATPase” a member of the HSP70 family of chaperones; requires ATP hydrolysis
Naked transport vesicles targeted to endosome…
Clathrin and adaptins recycled
See ECB figure 15-19
“Clathrin-coated pit”
To endosome…
Clathrin
Adaptin complexes
Lecture 17
PhagocytosisAutophagyEndocytosis
EndocytosisPinocytosis
Clathrin coated vesiclesReceptor-mediated endocytosis
Lipid micelle:
~800 phospholipids…
~500 molecules of cholesterol…
Example: Low-density lipoprotein (LDL), structure in which cholesterol is transported through our bodies
“Receptor-mediated endocytosis”
How do cells take up specific macromolecules?
Total mass: ~ 3 x 106 Da
~1500 molecules of cholesterol ester
1 copy of apoprotein B…
Overview of receptor-mediated uptake of LDL
Low pH of endosome (~6) causes LDL to dissociate from receptor
Receptor is recycled back to surface (cycles about every 10 min!)
LDL is transferred to lysosome (fusion of vesicles from TGN)
Hydrolytic enzymes cleave LDL, releasing cholesterol to cytoplasm for continued membrane biosynthesis in smooth ER
ECB 15-32
Defects in LDL endocytosis are associated with “familial hypercholesterolemia”…
–Severe atherosclerosis at early age (strokes and heart attacks in pre-teens)–Excess LDL in circulating blood–LDL not properly internalized by cells–Recessive/single gene… encoding plasma membrane receptor for LDL (LDL-receptor or LDL-R)
•Disease provided insight into mechanism of receptor-mediated endocytosis and identification/function of LDL-receptor
–Mutations in N-terminal domain: LDL-R doesn’t bind LDL…–Mutations in C-terminal domain: LDL-R is not internalized…
What does this tell you about function of domains of LDL receptor?
Domains in LDL receptor
Adaptin complex (four polypeptides)
Plasma membrane
Val
Tyr
Pro
Asn
LDL-R
LDL
Based on MBoC (3) figure 13-53
HOOC
N terminus of LDL receptor binds apoprotein B in LDL
C terminus binds adaptin
NH2
Recruitment of LDL-R to coated pits requires an “endocytosis signal” in cytoplasmic domain
Adaptin complex (four polypeptides)
Plasma membrane
Adaptin complex binds endocytosis signal in cytoplasmic domain of receptor:
-NPXY- (Asn-Pro-Val-Tyr) in LDL-R
At least three different adaptin complexes; bind different endocytosis signals on receptors
Adaptins recruit clathrin and initiate coated pit/vesicle formation
Val
Tyr
Pro
Asn
LDL-R
Endocytosis signal
LDL
Based on MBoC (3) figure 13-53
HOOC
A single coated pit has many different receptors and cargos
1,000s of receptors of many types per coated pit…
Same coated pits used for pinocytosis!
LDL-R
Low density lipoprotein (LDL)
Summary of “receptor-mediated” endocytosis of LDL
ATP
ADP+Pi
H+
Lysosome
Early endosome
ATP ADP+Pi
Uncoating(HSP70 family)
GTP
GDP+Pi
Coatedvesicle
Fusion(Snares)
Cholesterol ester cleaved
Cholesterol released for use
A single receptor makes hundreds of trips (~10 min/cycle)
Free cholesterol
pH ~7.2pH ~6
LDL-R
pH ~7-.7.2Low density lipoprotein (LDL)
Proton pump in endosome acidifies endosome lumen causing LDL to dissociate from receptor
dynamin
Coats for all reasons: a summary of vesicle coats and functions
COPs:Outbound: ER to Golgi transport, intra-Golgi, Golgi to plasma membraneRetrograde: intra-Golgi, Golgi to EREndosomal: early to late/lysosome
Clathrin:Plasma membrane to early endosome (endocytosis)Golgi to late endosome/lysosome
Don’t worry about COPI vs II
Endosomes sort internalized receptors and ligands
Transcytosis - movement of receptor to a different membrane from the one in which it was endocytosed
Some ligandsMany receptors
Many ligandsSome receptors
Maternal IgG–Secreted IgA–Others
ECB 15-33
“Transcytosis” moves maternal IgG across epitheliaIntestinal lumen
Apical membrane
Endosome
Endosome
Basolateral membrane
Milk duct
Maternal bloodNeonate blood
IgG is “secreted” across the mammary epithelium into milk by transcytosis
Receptor-mediated endocytosis from basolateral domain…
Secretion from apical membrane domain…
Epithelial cell
IgG in milk
IgG receptor
IgG receptor
Basolateral
Apical
IgG in blood
Tight junctions
IgG is transcytosed into the neonate blood Endocytosis from apical domain and secretion to basolateral membrane
Polarized epithelial cells have distinct apical and basolateral endosome compartments
Protein targeting and trafficking, finale!
Cytoplasm
Secretion/membrane proteins
Secretory vesicles
Lysosomes
Endosomes
RetrievalTransport
(constituitive secretion)
(regulated secretion)
Pro
tein
ta
rgeti
ng
Vesi
cle t
arg
eti
ng
RER
Golgi
Plasma membrane
Signal sequence
KDEL (soluble proteins)
KKXX (membrane proteins)
M6P
Nucleus NLS: (basic)
NES: (L-rich)
Mitochondria
Chloroplasts
Default
Signal peptide
Transit peptide
Endocytosis: From plasma membrane to endosome to lysosome…
PeroxisomesSKL at C term.
Endocytosissignal
ECB considers these all to be signal sequences
Membrane flow during exocytosis and endocytosis is a delicate
balance
Endocytosis internalizes membrane ~2-3% per minute…
Entire membrane is recycled in less than 1 hr…
Block endocytosis, exocytosis continues:
Block exocytosis, endocytosis continues:
Lysosome
ER
Golgi apparatus
Endosome
membrane area shrinks…
membrane area grows…
Original surface
Microfilaments: MuscleOrganelle transport in plants
Microtubules:Cilia and flagellaOrganelle transport in animals
Intermediate filaments:Cell structure
“Cytoskeleton”
ECB 1-20
Next lecture…