Module 1: The Endomembrane System III

Coated Vesicles

• vesicles with a protein coat on their cytosolic side

• Exocytosis: vesicles carry materials to the plasma membrane for secretion

• Endocytosis: vesicles internalize extracellular materials into the cell

5 Types of Coat Proteins

1. Clathrin

2. COPI

3. COPII

4. Caveolin

5. Caveolae

Clathrin

• coat protein involved in receptor-mediated endocytosis and vesicle transport from TGN to endosomes

COPI

• facilitates retrograde transport from the Golgi to the ER

Caveolin

• newly discovered coat protein found in vesicles of caveolae

COPII

• facilitates anterograde transport from the ER to the Golgi Complex

Caveolae

• specialized lipid rafts that are rich in cholesterol and sphingolipids

• these structures may be involved in cholesterol uptake in cells

The Function of Different Coat Proteins

1. helps determine the destination of the vesicle

   • clathrin directs vesicles to endosomes or plasma membrane

   • COPI facilitates retrograde transport (Golgi → ER)

   • COPII facilitates anterograde transport (ER→ Golgi)

2. induces curvature for the formation of the vesicles

   • clathrin forms lattice that helps shape the vesicle

   • dynamin assists in final pinching off of vesicles

3. prevents nonspecific fusion of the vesicles with another membrane

   • coat proteins provide selectivity in targetting with membranes

   • after uncoating, snare proteins ensure the vesicle fuses only with correct target membrane

Clathrin Coated Vesicles

vesicles are surrounded by lattice composed of:

1. Clathrin: provides structural support and bends membrane

2. Adaptor proteins: link clathrin to receptors

• the shape is the driving force to transform flat membrane into spherical vesicle

• essential for vesicle budding

Clathrin Triskelion

• basic unit of clathrin lattice

• multimeric protein composed of:

  • 3 heavy chains (form the outer framework of lattice)

  • 3 light chains (stabilize each leg internally)

    • radiates from central point

    • self-assemble into hexagons and pentagons to form curved lattice

Adaptor Protein Complexes in Calthrin Coated Vesicles

• AP complexes help assemble clathrin coats on vesicles

• act as a bridge between clathrin and membrane receptors

composed of:

• 2 adaptins: bind to cargo receptors

• 1 medium chain: recognize sorting signals

• 1 small chain: stabilize complex

• each subunit binds to different receptor, allowing the vesicle to selectively internalize specific molecules

How do clathrin coated vesicles form?

• AP complex binds to plasma membrane or TGN

• they concentrate receptors and cargo in specific regions

• ATP and GTP are required for coat assembly and vesicle budding

• clathrin assembles into hexagons, then pentagons as more clathrin triskelions are added (curvature induced)

• combination of hexagons and pentagons form spherical vesicle

• this curved coat enables vesicle to bud off the membrane

What is the role of dynamin and clathrin in vesicle formation?

Dynamin:

• GTPase that wraps around the neck of budding vesicle

• as GTP breaks down, dynamin constricts, causing the vesicle to pinch off from the plasma membrane or TGN

Clathrin:

• Clathrin forms the lattice coat that shapes the budding vesicle

• assembles into hexagons and pentagons to drive membrane curvature

Uncoating:

• after budding, the vesicle must shed its clathrin coat to fuse with target membrane

• clathrin rapidly dissociates

• requires energy from ATPase to dismantle the clathrin lattice

• this completes the target delivery

COPI-Coated Vesicles

• mediates retrograde transport

• Golgi → ER

• between Golgi cisternae for intra-Golgi transport

• maintains proper protein trafficking in all eukaryotic cells

Coat Components:

• COPI proteins: forms the lattice of vesicle coat

• ARF: small GTP binding protein that regulates coat assembly

  • facilitates recruitment of COPI proteins to membrane

  • after vesicle formation, ARF hydrolyzes GTP to GDP, leading to uncoating

How do COPI coated vesicles form?

1. ARF Activation

   • in the cytosol, ARF is inactive in its GDP bound form

   • at the membrane, a GEF triggers GDP → GTP exchange

   • this activates ARF causing conformational change that exposes hydrophobic tail

2. Coat Assembly

   • activated ARF-GTP recruits COPI coat proteins

   • COPI multimers assemble, causing membrane curvature and vesicle budding

3. Vesicle Uncoating

   • A GTPase-activating protein (GAP) stimulates GTP hydrolysis

   • ARF converts back to ARF-GDP

   • this triggers coat disassembly, making the vesicle ready for target fusion

COPII Coated Vesicles

• mediate anterograde transport from the ER to the Golgi

• essential for delivering newly synthesized proteins and lipids for further modification and sorting

Key Components:

• SAR1:

  • GTPase that regulates coat assembly/disassembly

  • functions like ARF in COPI

• Sec23/24 Complex:

  • selects cargo by binding to cargo receptors

• Sec13/31 Complex:

  • provides structural support

  • drives membrane bending and vesicle formation

How do vesicles eventually fuse with their target membranes?

• SNARE proteins ensure specific and correct fusion of vesicles with their correct target membranes

• prevents fusion with incorrect destinations

  1. v-SNAREs

     • located on the vesicles

     • recognize and bind to matching t-SNAREs

  2. t-SNAREs

     • located on target membranes

     • tether and fuse with v-SNAREs to enable membrane merging

v-SNAREs

• type of snare protein that ensures correct fusion of vesicles with their target membrane

• located on the vesicles

• recognize and bind to matching t-SNAREs

t-SNAREs

• type of snare protein that ensures correct fusion of vesicles with their target membranelocated on target membranes

• tether and fuse with v-SNAREs to enable membrane merging

SNARE Complex Dissembly

• Rab GTPases guide vesicles to the correct target membrane

• they help stabilize v-SNARE and t-SNARE pairing

• each target has specific Rab proteins

• after fusion, SNARE complexes must be dissembled for reuse

  • NSF is a chaperone that drives SNARE disassembly

  • SNAPs help recognize and bind to the SNARE complex

  • ATP hydrolysis drives the disassembly

Tethering Proteins

• help capture and position vesicles near their target membrane

• initiated before SNAREs

• explains why docking still occurs when SNAREs are blocked

2 Major Groups:

1. Coiled coil tethering proteins

   • recognize and tether COPI/COPII vesicles to the Golgi

   • function like long arms, pulling vesicles close to the membrane

2. Multisubunit protein complexes

   • key in protein secretion

   • guides vesicles to the plasma membrane for exocytosis

You are tracking vesicles by fluorescence and find that they are localized in the plasma membrane, trans Golgi and endosomes

1. They are coded with clathrin

2. They are coated with COPI only

3. They are coated with both COPI and COPII

4. They are coated with COPII only 

5. They lack dynamin

1. They are coded with clathrin

Lysosomes

• membrane bound organelles in the endomembrane system

• contain digestive enzymes capable of breaking down proteins, lipids, carbohydrates and nucleic acids

• contains acid hydrolases that function best in acidic conditions

• surrounded by a single membrane

• lumenal membrane is coated with glycoproteins to protect it from self-degradation

Types of Lysosomes:

1. Heterophagic

   • digest external materials brought in by endocytosis

2. Autophagic

   • degrade internal cell components (misfolded and damaged)

Lysosomes develop from endosomes

• lysosomal enzymes are synthesized in the rough ER

• modified and sorted in the Golgi

• packaged into vesicles and sent to endosomes

Endosome Maturation

1. Early Endosomes:

   • function as sorting stations for internalized material

   • start receiving digestive enzymes from the Golgi

   • pH at 6

2. Late Endosomes:

• accumulate enzymes and cargo for degradation

• cannot fuse with new vesicles as sorting ends

• acidification at pH 4-5, this is achieved by:

  • proton pumps V-ATPases

  • fusion with mature lysosomes

Lysosomes role in phagocytosis and receptor-mediated endocytosis

1. Phagocytosis:

   • phagosomes fuse with late endosomes or lysosomes

   • forms active lysosomes for digestion

   • enables phagocytes to break down pathogens

2. Receptor Mediated Endocytosis:

   • endocytic vesicles fuse with TGN vesicles carrying acid hydrolases

   • allows selective degradation of internalized receptor ligand complexes

3. Residual Bodies:

   • indigestible material remains in lysosomes as residual bodies

     • accumulates in others, contributing to cell aging

     • exocytosed in some cells

Autophagy

• breaks down damaged or unneeded cellular components

• recycles the resulting molecules for reuse

Link to disease:

• decreased autophagy is linked to increase tumour formation

Types of Autophagy:

1. Macrophagy

   • large scale degradation

   • target is enclosed in double membrane from ER, forming an autophagosome

   • autophagosome fuses with a lysosome

2. Microphagy

   • smaller scale degradation

   • material is directly engulfed by a single membrane before lysosomal degradation

Intracellular vs Extracellular Digestion

Intracellular Digestion:

• most lysosomal digestion occurs inside the cell

Involved in:

• phagocytosis

• receptor-mediated endocytosis

• autophagy

Extracellular Digestion:

• in some cases, lysosomes release enzymes outside the cell

• Ex: sperm cells release enzymes to break down barriers around the egg (fertilization)

• Ex: osteoclasts secrete enzymes to degrade bone tissue, regulating bone density

Lysosomal Storage Diseases

• caused by missing or defective lysosomal proteins

• lead to accumulation of undigested substances

Examples:

• Type II glycogenosis: buildup of glycogen

• Hurler & Hunter syndromes: buildup of glycosaminoglycans

• Tay-Sachs disease: buildup of gangliosides in the nervous system

Peroxisomes

• single membrane bound organelles

• not derived from the ER and not apart of the endomembrane system

• self-replicating

Functions of Peroxisomes

• contains oxidative enzymes for metabolic processes

• houses hydrogen peroxide generating reactions

• contains catalase, which breaks down hydrogen peroxide

• in animals, contains urate oxidase

• in plants, contains crystalline catalase

• many peroxisomes have crystalline enzyme cores

Essential Functions of Peroxisomes

1. Hydrogen peroxide metabolism:

   • oxidases generate it during metabolic reactions

   • catalase detoxifies it by converting it into water and oxygen

   • protects cells from oxidative stress

2. Detoxification of harmful compounds:

   • reactive oxygen species are neutralized

   • superoxide dismutase converts ocygen into hydrogen peroxide, then degraded by catalase

   • prevents damage to proteins and lipids

3. Fatty acid oxidation:

   • involved in beta oxidation of long fatty acids

   • animals produce acetyl CoA which is used for biosynthesis

   • plants and yeast fully oxidize fatty acids within peroxisomes

4. Nitrogen metabolism:

   • in most animals, peroxisomes break down urate

5. Catabolism of unusual substances:

   • degrade D-amino acids

   • breaks down xenobiotics

   • helps protect the cell from toxic buildup

Animal Peroxisomes Disorders

• many disorders result from defective peroxisomal proteins

• most common is X-ALD

• caused by defect in transporter protein that moves long chain fatty acids into peroxisomes for degradation

• leads to accumulation of toxic fatty acids

Plant Specific Peroxisomes

• work with mitochondria and chloroplast to regulate energy metabolism

  1. Leaf Peroxisomes:

     • found in photosynthetic tissues

     • involved in the photorespiratory pathway

  2. Glyoxysomes:

     • found in plant seeds

     • helps convert stored lipids into sugars during germination

     - both help recover carbon lost during photorespiration

Peroxisome Biogenesis

peroxisomes increase via:

1. Division of existing peroxisomes

   • regulated by dynamin-related proteins for membrane fission

2. De novo formation from vesicles budding off the ER or Golgi

   • these vesicles carry membrane and matrix proteins

   - the formation of new peroxisomes require PEX proteins

You are studying mutant cells that no longer contain acid hydrolases. Which organelles are these cells defective or deficient in?

1. Lysosomes and peroxisomes

2. Peroxisomes and endosomes

3. Peroxisomes

4. Lysosomes, peroxisomes, and mitochondria

5. Lysosomes

1. Lysosomes