Vesicle formation and targeting is a multi-step process 3. Targeting and docking to specific compartment… 2. Formation of coated transport vesicle… GAP.

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)

Pro

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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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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

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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…