Endocytosis

What is the general pathway of the endocytic pathway?

1. cell surface

2. early endosome

3. late endosome

4. lysosome

What is the structure of clathrin?

• triskelion

  • 3 heavy and 3 light chains

  • forms a lattice and induces membrane curvature

• clathrin is very versatile

  • many types of vesicles that recruit different cargos on different receptors and moves them to different destinations

Does clatherin bind cargo?

no - it requires adaptor proteins

What are the main clatherin adaptors?

1. AP-2 - major plasma membrane adaptors

2. ARH - specific to LDL receptors

one side binds clatherin and the other side binds a cargo receptor

What is used to bud off clathrin vesicles?

Dynamin

• a GTPase

• forms a helix around neck of the vesicle

mechanism: GTP hydrolysis → constriction → scission (pinches off vesicle)

what are the characteristics of the early endosome?

• first compartment

• mildly acidic

• has tubular and vesicular regions

• function: sorting station

• at this low pH the ligand detaches from the receptor

  • ligand takes path to lysosome

  • receptors are sorted into tubular regions which bud off and become recycling endosomes

• marked by Rab5

• EEA1 is involved in vesicle docking/fusion

what are the characteristics of the recycling endosome?

• derived from early endosome tubules

• less acidic

• returns membrane and most receptors to the cell surface

• marked by Rab11

what are the characteristics of the late endosome?

• more acidic

• contain internal vesicles (inside the lumen)

• formed by inward budding of membrane proteins into vesicles

• pre-lysosomal function → prepares cargo for lysosomal degradation

• DO NOT contain recycling receptors because cargo is committed to degradation

• marked bt Rab7

  • can recruit HOPS complex for tethering and fusion

  • motor proteins dyenin and kinesin

what are the characteristics of the lysosome?

• highly acidic (pH = 5 or lower)

• acidic environment for enzyme activity

• contains many enzymes including (cathepsin D, acid phosphatase, saposins)

• if lysosome ruptures the enzymes it contains become inactive in the cytosol (prevent damage)

• enzymes could digest the compartment itself

  • solution: lysosomal membrane contains LAMPS and LIMPS which are highly glycosylated protective barrier

• marked by Rab7

What is the endocytosis of LDL pathway?

• LDL delivers cholesterol and lipids from blood to cells

Outer Layer:

• phospholipid monolayer

• ApoB protein which is a binding site for receptor

Core:

• cholesterol esters

• triglycerides

• some free cholesterol

Key idea: LDL is basically a hydrophobic lipid package made soluble in the blood

Why do cells take up LDL?

cells need cholesterol for membranes and synthesis

• if low on cholesterol → up regulate LDL receptor expression

What are the steps of the endocytosis of LDL pathway?

1. LDL binds receptor (at neutral pH)

2. LDL receptor is specifically recruited into clathrin coated pits

3. vesicle forms and LDL enters the cell

4. gets to early endosome where pH drops → LDL dissociates from receptor

5. receptor is retuned to plasma membrane via tubular regions

6. LDL particle stays in lumen → degradation

7. once in lysosome LDL is broken down and everything is used by the cell

What is the difference between inulin and LDL?

Inulin:

• endocytosed and degraded

• purpose: single termination

LDL:

• endocytosed and degraded

• purpose: nutrient delivery

What is the EGF receptor pathway?

• function: growth signalling (cell division)

• receptor is degraded in this case NOT recycled

STEPS:

1. EGF binds to EGFR (receptor)

2. High EGF → receptor dimerizes and becomes ubiquitinated

3. endocytosis

4. early endosome - EGF stays bound to receptor

5. receptor sorted into MVB

6. lysosome → degradation

What is the shut off mechanism for the EGF pathway?

• once the receptor is inside internal vesicles, it cannot signal anymore

• it is degraded and permanently shut off

• if there is low EGF the receptor may be recycled to maintain low-level signalling

What is transferrin?

• RNA binding protein

• can be floating in bloodstream and between cells in your body

What is the Transferrin pathway?

• function: bring iron into cells (Fe3+)

• essential for processes like hemoglobin synthesis

• a cell that needs iron will express the receptor on its surface

STEPS:

1. holo-transferrin binds to receptor

2. transferrin and the receptor internalized via clatherin

3. in early endosome (acidic pH=5.5) → transferrin stays on the receptor but the iron comes off

4. Fe3+ is converted to Fe2+ via STEAP3

5. Fe2+ exported to cytoplasm via DMT1

6. transferrin and the receptor go back to the surface together

7. at cell surface (neutral pH) → apo-transferrin detaches from the receptor

What is the transcytosis pathway?

• transport across a cell → endocytosis then exocytosis on a different side

• happens in polarized epithelial cells

• the problem the body is solving: transport of antibodies

  • Antibodies (IgA) are made by plasma cells (NOT epithelial cells)

  • but they are needed in the lumen → cell must move antibodies across itself

STEPS:

1. binding on basolateral side of the IgA to pIgA (receptor)

2. internalized via clatherin-mediated endocytosis

3. moves through endosomal system

4. delivery to apical surface

5. receptor is cleaved

6. IgA released into lumen as secretory IgA (sIgA)

• IgA ends up in mucus/ lumen

• binds pathogens to prevent infection at surfaces

What is caveolae transcytosis pathway?

• not normal endocytic pathway → no interaction with endosomes

• small, flask shaped pits

• made of calvolin

• cholesterol rich

• forms a hairpin in membrane

• calveolae cannot uncoat like clatherin vesicles

• happens in endothelial cells (thin and move things across quickly)

• albumin can be transported

STEP:

1. caveola forms at plasma membrane

2. pinches off (dynamic required)

3. moved short distance across cell

4. fuses with opposite membrane

What is the pro collagen experiment?

• procollagen can only leave the ER when vitamin C is present

• add vitamin C briefly → release a pulse of cargo

• observation:

  • cargo appears: in cis → medial → then trans

  • BUT: never spreads across all cisternae

  • interpretation is that cargo as a coherent unit

What was the reasoning for if cisternae move?

1. COP I transport the enzymes backwards to get them in the right spot

2. Golgi has tubular connections where enzymes can diffuse between cisternae, then they accumulate where their substrates are

What are the two types of secretion?

1. Constitutive Secretion

• occurs continuously in all cells

• vesicles → fuse immediately with plasma membrane

• no signal required

• deliver membrane proteins and supply lipids to the membrane

2. Regulated Secretion

• occurs only in specialized cells

• vesicles are stored in the cytoplasm

• requires a signal

• ie. hormones, digestive enzymes, mucus, neurotransmitters

What happens in polarized cells?

• apical surface and basolateral surface are separated by tight junctions

• proteins cannot diffuse between domains

• the only way to move between the two surfaces are endocytosis → transcytosis

• sorting matters because there are different proteins that must go to different sides

  • apical → ion channels, secretion

  • basolateral → receptors (LDL, transferrin)

• basolateral side faces the blood while the apical side faces the lumen

What is the apical secretion pathway?

• uses tubular transport intermediates

• no clathrin coat

STEPS:

1. Lipid rafts form in TGN

2. Proteins partition into rafts

3. Kinesin pulls membrane → forms tubule

4. Dynamin-like proteins → fission

5. Tubule moves to apical surface

• sorting mechanism is based on membrane properties → lipid rafts (cholesterol + sphingolipids) - thicker and more rigid

  • long transmembrane domains → raft → apical

  • short transmembrane → non-raft → basolateral

What are the apical targeting signals?

• GPI anchor (strong signal)

• lipid raft association

• O-linked glycosylation

• N-linked glycosylation (weak)

Which signal overrides another?

• basolateral signals OVERRIDE apical ones

describe the example of the GPI anchored proteins

• GPI proteins strongly prefer lipid rafts

• lipid rafts are enriched in cholesterol and sphingolipids

• these proteins are therefore sorted into raft domains in the TGN

• raft domains then cluster in the TGN → pulled into tubular carriers → tubules move via kinesins → delivered to apical membrane

What is process for basolateral secretion?

• proteins destined for the basolateral membrane are sorted at the TGN

• basolateral signalling signals are located in the cytoplasmic tail of transmembrane proteins

• sorting signals:

  • tyrosine based motifs → y-x-x-bulky hydrophobic residue or NPxY

  • dileucine motifs → [DE]xxxLL

• most basolateral proteins are transported in clatherin-coated vesicles → forms outer coat and drives vesicle budding from TGN

What is the role of adaptor protein AP1B?

• specific adaptor for basolateral sorting

• its role is to recognize tyrosine motifs and dileucone motifs

• links cargo to clatherin

• two versions:

  • AP1B → basolateral surface

  • AP1A → endosomes

What are all the versions of the AP family?

• AP1B → basolateral membrane (has mu1-b)

• AP1A - endosomes (has mu1-a)

• AP2 → endocytosis

• AP3 → lysosomes

• AP4 → not covered

• same clatherin but different inner adaptor → giving specificity

What are the two pathways to the lysosome?

1. direct pathway (main)

• TGN → endosome → lysosome

• used for mostly lysosomal enzymes (hydrolases)

• efficient and targeted

• uses sorting receptors

2. indirect pathway

• TGN → plasma membrane → endocytosis → lysosome

• less direct

• involves re-endtry vis endocytosis

• uses endocytosis signals

what are the types of proteins found in lysosome?

A. catabolic proteins (breakdown enzymes) → ie. saposins

B. protective membrane proteins → heavily N-glycosylated → ie. LAMPS

What are the types of lysosomal proteins and what signals are used?

• signals:

  • tyrosine based motifs → y-x-x-bulky hydrophobic residue

  • dileucine motifs → DxxLL or [DE]xxxLL[LI]

• motifs interact with GGA or AP3 complex

• A. soluble enzymes in the lumen → need cargo receptors and cannot bind coat directly

• B. membrane proteins → can be sorted directly and use AP3, GGA and AP1A

What is the mannose 6 phosphate system?

• maintains targeting signal for lysosomal enzymes

• happens in the golgi on N-linked oligosaccharides

• lysosomal enzymes already have sugars on the but we are modifying those sugars to add a tag

what is the actual mechanism/ steps for the M6P system?

1. add GlcNAc-phosphate → GlcNAc phosphotransferase adds GlcNAc phosphate onto a mannose sugar

2. removal of GlcNAc → leaves behind M6P which is the targeting signal

How does the enzyme actually get to the lysosome?

1. M6P binds to the receptor M6PR

2. complex is packaged into clatherin coated vesicle with he help of GGA and AP1

3. vesicle binds off from TGN → loses coat and fuses with endosome

4. in endosome pH is more acidic (pH=5) so the M6P receptor releases enzymes

5. enzyme stays in endosome → lysosome to be degraded

6. receptor is sent back to Golgi via the retromer pathway

What is a lysosomal storage disease?

• lysosome cannot degrade materials causing accumulation and enlarged lysosomes

What is I-cell disease?

• cause: mutation in GlcNAc phosphotransferase → responsible for adding the M6P tag

• without a tag lysosomal enzymes cannot be recognized

• therefore they do not bind M6P receptor and do not enter clatherin vesicles to endosomes

• instead they follow the default pathway through the secretory pathway

• in the cell since the lysosome is missing its hydrolases it cannot degrade material → causes build up of proteins, lipids and sugars = large swollen enzymes

• causes developmental defects

• HOWEVER some enzymes still reach the lysosomes so there must be another targeting pathway → sortilin pathway

What is the GGA adaptor protein?

has 4 domains:

• VHS: recognizes and binds to both forms of M6PR and sortilin

• GAT: binds to Arf1-GTP

• Hinge: binds clathrin

• GAE: binds to accessory proteins

GGA is a single polypeptide protein

GGA does not bind enzymes directly → it binds sorting receptors

What are the roles of Arf1?

a small GTPase

• when GTP-bound → active and recruits GGA to the membrane

• used in COPI vesicle pathway, GGA recruitment and AP adaptor recruitment

describe the interaction of GGA and AP1

• transport to endosome

What is the sortilin pathway?

• sortilin does not require a M6P tag

• binds cargo directly

• example of cargo = saposins → reach lysosomes even in patients with I-cell disease and helps degrade sphingolipids

  • without proper activation of saposins, lipid breakdown is inefficient

• act as solubilizers and liftases

• transport uses GGA and AP1

What is gaucher disease?

• mutation in enzyme beta-glucocerebrosidase

• normally this enzyme breaks down a lipid. into ceramide and sugar

• if the enzyme is missing or defective, glycolipisa accumulate inside lysosomes

• matters the most in macrophages which are constantly engulfing and breaking down membranes which are rich in lipids

• specifically glycolipids that accumulate

• consequences: lysosomes swell and cells become dysfunctional

describe the GGA mutant experiment

• they removed the clathrin binding domain

• result: GGA still binds receptor but cannot form a vesicle

• consequences:

  • receptors get stuck in golgi

  • cargo does not get to lysosome → rather secreted outside the cell

What happens if you knock out AP3?

LAMPS go to plasma membrane instead of lysosome

What is phagocytosis?

• Uptake of large particles

• done only by specialized cells:

  • macrophages

  • neutrophils

  • dendritic cells

• uses large vesicles → phagosomes

• requires actin remodelling and receptor binding

What is pinocytosis?

• cell drinking

• occurs in almost all cells

• takes in fluid and small dissolved particles

• uses small vesicles

• includes clathrin dependent and independent pathways

how does phagocytes recognition occur?

• particles are coated with antibodies

• phagocytes have Fc receptors

• Fc receptors bind constant region of antibodies

• antibodies act as tags

Describe the zipper model of phagocytes

• binding occurs progressively

• membrane wraps around the particle like a zipper

• requires continuous receptor engagement

• if antibodies are unevenly distributed → phagocytosis fails

What are the steps of mechanism for the zipper model?

1. antibody coated particle binds Fc receptors

2. actin polymerization begins

3. pseudopods extend

4. particle is enclosed → phagosome formed

what are the properties of the zipper model?

properties:

• actin dependent

• pseudopod formation

• zipper-like engulfment

What is the role of actin?

required for membrane extension

driven by Rho GTPases

required for final enclosure

What are the steps for phagosome maturation?

1. fusion with early endosome: gains Rab5

2. fusion with late endosome: gains Rab7

3. fusion with lysosome → phagolysosome

4. phagolysosome is degradative

what are the characteristics of the phagolysosome?

• highly acidic

• contains degradative enzymes

• kills most bacteria

How can pathogens evade the phagolysosome?

• block maturation

• stop Rab5 → Rab7 transition

• prevent acidification

• survive inside macrophages

What is the complement system?

• activated by antibodies

• can punch holes in bacteria and bind randomly to surfaces

• can kill bacteria directly

What is autophagy?

• self-eating

• cell takes cytoplasmic material → delivers to lysosome → degrades

• enters the endocytic pathway internally

• highly regulated → targets specific structures

Why do cells use autophagy?

• starvation → nutrients

• remove defective organelles

• remove protein aggregates from cytoplasm

• remove pathogens

• lipid metabolism: release cholesterol from lipid droplets

What is mitochondrial specific autophagy?

• defective mitochondria must be removed

• they have their own DNA so mutations can arose

• failure to remove them can result in parkinson’s and neurodegeneration

Three types of autophagy

1. macroautophagy

• large structures

• double membrane organelle

2. Chaperone mediated autophagy

• proteins tagged with KFERQ sequence

• binds HSC70

• enters lysosome via LAMP2A pore

• very selective and limited

3. microautophagy

• cytoplasm directly buds into late endosome

• involves Hsc70 + phosphatidylserine

• minor pathway

What is the mechanism for macroautophagy?

1. C shaped membrane has autophagy receptors

2. cell does not engulf randomly → ubiquitin tags cargo → p62 binds ubiquitin → LC3 binds p62

3. LC3 is cleaved → LC3II → conjugated to PE

4. phagophore grows around the cargo → encloses it

5. vesicle is sealed with a double membrane

6. autophagosome interacts with early endosomes and late endosomes

7. once fused with lysosome it forms an autophagolysosome and the contents are degraded

*NOTE: inner membrane is degraded too → LC3 is not recycled, its degraded

What are Rab proteins?

• master regulators of membrane trafficking

• each Rab controls one specific trafficking step

• defines organelle identity

• small GTPases

What are effectors?

• there are multiple effectors per Rab

• types:

  • tethers

  • snares

  • motor proteins

  • lipid modifying enzymes

What is the relationship between Rab and GDI?

• GDI extracts Rab-GDP from the membrane

• hides the lipid tail

• returns the Rab to the cytosol

• there are only a few GDI for many Rabs

What are tethering proteins?

• examples:

  • EEA1 → Rab5 (early endosome)

  • p115 → Rab1 (ER → Golgi)

• function:

  • it recognizes the correct target membrane

  • brings membranes close together before fusion

What are regulators of motor proteins?

• function: control movement of vesicles and organelles

• examples:

  • Rab7 recruits RILP → dyenin and FYCO1 → kinesin

What are regulators of lipid metabolism?

• function: modify lipid membrane lipids → define organelle identity

• example: Rab5 recruits PI3-kinase → produces PI3P

• lipids then act as signals for protein recruitment