Module 1: The Endomembrane System II

Endomembrane System

• network of interconnected organelles of a eukaryotic cell

• this system coordinates the synthesis and processing of lipids and proteins

• involved in trafficking of materials to maintain cellular function

• ER; site for protein (rough ER) and lipid synthesis (smooth ER)

• Golgi complex: responsible for further processing, modifying and sorting of proteins and lipids received from the ER

Roles of ER and Golgi in protein and lipid trafficking

1. Protein Targetting and Retention

   • Proteins made in the rough ER are directed to their proper destinations or for secretion.

   • Tags ensure correct delivery and retention:

     • Amino acid sequences (e.g., KDEL for ER retention)

     • Hydrophobic domains (for membrane localization)

     • Oligosaccharide chains (for targeting/modification)

   • Tags can also prevent incorrect packaging into vesicles

2. Membrane Lipid Tagging

   • Lipids are tagged (often with phosphate groups) for correct vesicle targeting.

   • These tags guide lipids to destinations like the plasma membrane, lysosomes, or endosomes.

   → Overall: Proper tagging ensures accurate sorting and trafficking, preventing errors that can disrupt cell function or cause disease.

Mechanisms for Protein Trafficking

1. Retrieval Mechanism

   • brings back misdirected proteins to the ER

2. Retention Mechanism

   • keeps proteins in their proper compartments (ER or Golgi)

   • the TGN is final sorting station in the Golgi complex, sending proteins to:

     • The plasma membrane (for secretion or membrane insertion)

     • Lysosomes or other organelles

     • Secretory vesicles for exocytosis

What is the role of the ER in Protein-Trafficking?

1. Retention in the ER:

   • ER specific proteins are kept in the ER using retention tags, like RXR

   • these tags prevent escape and ensure proteins are properly folded before leaving

   • example: NMDA receptor has an RXR tag that keeps subunits in the ER until the full complex is assembled

   • once assembly is completed, the tag is masked, allowing export

2. Retrieval to the ER:

   • Proteins mistakenly sent to the Golgi are retuned to the ER using retrieval tags

   • retrieval tags at the C-terminus include:

     • KDEL or KKXX in mammals

     • HDEL in yeast

   • these tags bind receptors in the Golgi, triggering formation of vesicles that return the proteins to the ER

Overall function:

• maintains correct protein localization in the ER

• prevents trafficking errors

What is the role of the Golgi in protein trafficking?

1. Retention Tags:

   • some Golgi proteins have tags that prevent them from leaving the Golgi

   • these tags help maintain the correct protein composition within each Golgi compartment

2. Retrieval Tags:

   • other proteins have retrieval tags that allow them to be returned to the Golgi if they exit by mistake

   • these work similarly to ER retrieval tags (like KDEL)

3. Size-based Exclusions:

   • large protein complexes that cannot fit into vesicles are excluded from transport, to keep the golgi in place

4. Membrane Thickness Matching:

   • the Golgi membrane thickens from cis → trans

   • proteins are retained based on their domain length:

     • short domain length → stays in cis-Golgi

     • long domain length → stays in trans-Golgi

Hydrophobic Domain Length within the Golgi

• Golgi proteins are membrane proteins with hydrophobic transmembrane domains

• membrane thickness increases along the secretory pathway (ER→ Golgi → plasma membrane)

• as proteins move forward, they are retained when the membrane becomes too thick for their hydrophobic domain to span

• this traps proteins into the correct compartment

• shorter domain → ER or cis-Golgi

• longer domain → trans-Golgi or plasma membrane

Protein sorting in the TGN

• lysosomal enzymes undergo N-glycosylation (removal of glucose and mannose units) in the ER and early Golgi

• in the Golgi, mannose residues are phosphorylated, forming a mannose-6-phosphate (M6P) tag

• this M6P tag acts as a signal to direct these enzymes from the TGN to the lysosomes

How are KDEL tags similar to mannose-6-phosphate tags?  

1. They are added to proteins in the Golgi network

2. They are removed from proteins once the proteins get to their proper destination

3. They bind to specific receptors, which then target them to specific intracellular compartments

4. They are amino acid sequences that cells use to sort proteins to specific intracellular compartments

5. All of the above

1. They bind to specific receptors, which then target them to specific intracellular compartments

Formation of Mannose-6-Phosphate (M6P)

1. Formation of M6P Tag:

• Phosphotransferase (in early Golgi) adds GlcNAc-1-phosphate to mannose residues.

• In the mid-Golgi, GlcNAc is removed, leaving behind mannose-6-phosphate (M6P).

2. Role of the M6P Tag:

• Acts as a signal for targeting lysosomal enzymes.

• Ensures proteins are recognized by M6P receptors (MPRs) in the trans-Golgi network (TGN).

• MPR-protein complexes are packaged into vesicles and transported to endosomes.

3. Transport Pathway:

• Vesicles from the TGN deliver enzymes to late endosomes.

• These enzymes eventually reach lysosomes for degradation functions.

Multi-vesicular Endosomes (MVEs/MVBs)

• Specialized late endosomes that:

  • Degrade damaged proteins, membrane receptors

  • Recycle useful components

• Serve as an intermediate step between early endosomes and lysosomes.

Lysosomal Enzyme Relating Disorders

I-Cell Disease (Inclusion-Cell Disease):

• Caused by a defect in phosphotransferase, the enzyme that adds the M6P tag.

• Without M6P, enzymes are not targeted to lysosomes and are secreted outside the cell.

• This leads to waste buildup in cells and impaired degradation, causing severe developmental issues.

Lysosomal Enzyme Sorting

• Lysosomal enzymes dissociate from M6P receptors (MPRs) in the late endosome.

• This prevents enzymes from being sent back to the Golgi.

• MPRs are recycled to the Golgi for reuse.

• The late endosome matures into a lysosome or merges with an existing one.

• This ensures proper breakdown of waste, organelles, and macromolecules

How do vesicles transport materials across the plasma membrane?

1. Exocytosis

   • vesicles fuse with the plasma membrane

   • releases contents outside the cell

   • also delivers lipids and proteins to the membrane

2. Endocytosis

   • plasma membrane folds inward to form vesicles

   • internalizes nutrients, signals or other substances

   • involved in recycling membrane components

Secretory Pathways

• transport molecules, especially proteins from ER site of synthesis to outside of the cell

Steps in Secretory Pathways

1. ER

   • proteins are synthesized and folded in the rough ER

2. Golgi Complex:

   • proteins are modified, sorted and packaged into vesicles or granules

3. Vesicles/Granules:

   • vesicles accumulate near the plasma membrane

   • when signalled, they fuse with the membrane

4. Exocytosis

   • vesicle contents released outside of the cell, completing secretion

How does Exocytosis work?

1. Constitutive Secretion

   • unregulated and continuous

   • vesicles from TGN fuse with plasma membrane

   • delivers proteins and lipids

   • once was thought to be default pathway (now tagging required)

2. Regulated Secretion

   • vesicles accumulate in the cell and wait for a specific signal to trigger release

   • ex: neurotransmitter release in nerve cells

   • vesicles form in the TGN and undergo maturation

     • protein condensation

     • proteolytic processing (activate enzymes/hormones)

   • once maturated, vesicles move near the membrane, signalled to fuse together and release contents

3. Polarized Secretion

   • exocytosis occurs only on a specific side of the cell

   • common in polarized cells like epithelial cells

Exocytosis in animal vs plant/fungal cells

Animal cells:

• secrete hormones, mucus, milk proteins, and digestive enzymes

Plant/fungal cells:

• secrete enzyme and structural proteins for the cell wall

Process of Exocytosis

1. Vesicles from the Golgi complex carry secretory products to the plasma membrane

2. Vesicle membrane fuses with the plasma membrane, forming a continuous bilayer

3. Contents are released outside the cell

4. Vesicle membrane becomes part of the plasma membrane

   • The inner (lumenal) side of the vesicle becomes the outer surface of the membrane

   • Glycolipids and glycoproteins made in the ER/Golgi now face outside the cell

Neurotransmitters are released only on one side of a nerve cell and they depend on external signals before secretion. This is ____. 

1. Constitutive polarized secretion

2. Constitutive non-polarized secretion

3. Regulated polarized secretion

4. Regulated non-polarized secretion

5. None of the above

1. Regulated polarized secretion

Constitutive Secretion

• unregulated and continuous

• vesicles from TGN fuse with plasma membrane

• delivers proteins and lipids

• once was thought to be default pathway (now tagging required)

Regulated Secretion

• vesicles accumulate in the cell and wait for a specific signal to trigger release

• ex: neurotransmitter release in nerve cells

• vesicles form in the TGN and undergo maturation

  • protein condensation

  • proteolytic processing (activate enzymes/hormones)

• once maturated, vesicles move near the membrane, signalled to fuse together and release contents

Polarized Secretion

• exocytosis occurs only on a specific side of the cell

• common in polarized cells like epithelial cells

How does Endocytosis work?

1. plasma membrane folds inward, keeping extracellular material

2. the membrane pinches off, forming an endocytic vesicle

3. vesicles matur into an early endosome

4. this endosome fuses with vesicles from the trans-golgi network

5. these TGN vesicles deliver digestive enzymes

6. early endosomes mature into late endosomes, then into lysosomes, which degrade the ingested material

Membrane Flow during Endocytosis and Exocytosis

• Exocytosis adds lipids and proteins to the plasma membrane

• Endocytosis removes lipids and proteins from the plasma membrane

• both work in opposite direction to regulate membrane flow

What are the 3 Different Types of Endocytosis?

1. Phagocytosis

2. Receptor-mediated endocytosis (clathrin dependent)

3. Clathrin-independent endocytosis

Pinocytosis

• cells engulf liquids or small molecules

• type of endocytosis

Phagocytosis

• type of endocytosis

• used in unicellular organisms for feeding

• humans (multicellular) are limited to specialized cells called phagocytes

• Phagocytes include neutrophils and macrophages

  • function: destroy pathogens (immune defense) and clear debris (tissue repair)

Process of Phagocytosis

1. particles trigger pseudopod formation (membrane extensions)

2. pseudopods engulf the particle, forming a phagosome

3. the phagosome fuses with a lysosome or late endosome, forming a phagolysosome

4. enzymes inside break down the contents

5. produces toxic oxidents like hydrogen peroxide and superoxide kill pathogens

Receptor-Mediated Endocytosis (Cathrin Dependent)

• selective endocytosis process

• cells internalize specific macromolecules by binding them to receptors on the plasma membrane

• used to uptake molecules like hormones, growth factors, LDL cholesterol

Process of Receptor-Mediated Endocytosis (Cathrin Dependent)

1. Ligand binding to receptors:

   • ligands (LDL, hormones) bind to specific receptors on cell surface

2. Movement to coated pits:

   • receptor-ligand complexes move into clathrin coated pits

3. Adaptor proteins:

   • clathrin forms lattice that shapes the membrane into pit

   • dynamin (GTPase) pinches off the vesicle from plasma membrane

4. Formation of clathrin coated vesicle:

   • a clathrin coated vesicle forms, enclosing the ligand-receptor complex

5. Uncoating of the besicle:

   • the clathrin coat is removed, and coat proteins are recycled

6. Fusion with early endosomes:

   • uncoated vesicle fuses with early endosome where sorting occurs

     • Recycling

     • Degradation

7. Endosome maturation:

   • early endosome retain enzymes from TGN and mature into late endosomes, then lysosomes

   • lysosomes degrade the internalized material

Pathways for Ligand-Receptor Complex in Receptor-Mediated Endocytosis

• After entering the early endosome, ligand-receptor complexes can follow one of several paths:

  1. Degradation in Lysosomes (7a):

  • Sent to late endosomes → lysosomes

  • Both ligand and receptor are broken down by enzymes

  • Recycled as basic molecular components

  2. Recycling to the Plasma Membrane (7b):

  • Receptors dissociate from ligands in the acidic early endosome

  • Receptors are returned to the membrane for reuse

  3. Transcytosis (7c):

  • Ligand-receptor complexes are sent to a different area of the plasma membrane

  • Allows direct transport across cells, e.g., in epithelial cells or the blood-brain barrier

  4. Delivery to the Trans-Golgi Network (TGN):

  • Some complexes are routed to the TGN

  • Used for further sorting, modification, or redistribution within the cell

These cellular components are recycled following receptor-mediated endocytosis except?

1. The ligand

2. The receptor

3. Adaptor protein

4. Clathrin

5. Dynamin

1. The ligand

Clathrin-Independent Endocytosis

• nonspecific uptake of extracellular fluid and dissolved solute

  (bulk-phase endocytosis)

• internalized material and fluid are delivered to early endosomes for sorting

• operates continuously (constant rate) for steady membrane turnover

• balances membrane flow by compensating for exocytosis

Bulk-Phase Endocytosis

• type of pinocytosis

• apart of clathrin-independent endocytosis

• nonspecific internalization of extracellular fluid and dissolved solutes

• does not require receptors or ligand binding

• Unlike receptor-mediated endocytosis, it does not concentrate specific molecules