Module 1: The Endomembrane System I

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

Components of the Endomembrane System

• rough ER

• smooth ER

• golgi complex

• endosomes

• lysosomes

Rough ER

• site of protein synthesis and initial modifications

• has ribosomes attached to cytosolic side, giving rough appearance

• forms transition vesicles to shuttle lipids and proteins from ER to golgi complex

Smooth ER

• site of lipid synthesis and detoxification

• lacks ribosomes

Endosomes

• sorting hubs for materials brought into the cell via endocytosis

• directs these materials to the lysosomes or other destinations

Lysosomes

• contains enzymes to digest ingested materials and degrade damaged cellular parts via autophagy

Endoplasmic Reticulum (ER)

• continuous network of flattened sacs, tubules and vesicles that extend throughout the cytoplasm

• contains ER cisternae and ER lumen

ER cisternae

• membrane bound sacs that make up the ER

ER lumen

• internal space within the cisternae, where biological processes take place

Functions of the rough ER

makes proteins for:

• Plasma membrane (e.g., receptors, transporters)

• Endomembrane organelles (e.g., lysosomes, endosomes)

• Secretion outside the cell (e.g., hormones, enzymes)

Functions of the smooth ER

Produces lipids, including:

• Phospholipids (for membranes)

• Steroids (e.g., cholesterol, hormones)

• Triglycerides (for energy or membrane building)

Difference Between Rough and Smooth ER

Rough ER:

• forms large flattened sheets

With exception of:

• transitional elements resemble tubular structure of smooth ER

• lumenal spaces of both ER are continuous, allowing for material and signal flow between both regions

Smooth ER

• forms tubular structures

what is the variation in amount of rough ER and smooth ER

1. cells with rough ER networks are involved in synthesis of secretory proteins

2. cells with extensive smooth ER are specialized in production of steroid hormones (lipids)

Main Function of Rough ER

• involved in protein synthesis and processing

• the ribosomes attached to cytosolic side are responsible for synthesizing proteins

the types of proteins synthesized in rough ER

Membrane bound proteins:

• Become part of the endomembrane system

Soluble proteins:

• Function within the lumen of organelles

• Or to be secreted out of the cell

Co-Translational Insertion

• As the ribosome makes a protein, the growing polypeptide is inserted into the endomembrane system via a pore in the rough ER membrane

• This process makes sure that proteins are correctly inserted into the ER lumen during synthesis

Other functions of the rough ER

• glycoprotein formation

• folding of polypeptides

• removal of defective or misfolded proteins

• assembly of multimeric proteins

Functions of Smooth ER

• drug detoxification

• lipid biosynthesis

• calcium storage

• carbohydrate metabolism

How does drug detoxification work in smooth ER?

• Hydroxylation adds hydroxyl (-OH) groups to hydrophobic drugs/toxins
  → Increases water solubility for easier excretion

• Cytochrome P-450 enzymes (monooxygenases) catalyze these reactions

• Aryl hydrocarbon hydroxylase (a P-450 enzyme) breaks down carcinogenic polycyclic hydrocarbons

How does carbohydrate metabolism work in smooth ER?

• In liver cells, glycogen is stored as granules near the smooth ER

• glycogen is broken down into glucose-6-phosphate

• Glucose-6-phosphatase removes the phosphate to form free glucose

• Free glucose is released into the bloodstream to be used for energy by other tissues

How does calcium storage work in smooth ER?

• Sarcoplasmic reticulum (a type of smooth ER in muscle cells) regulates calcium levels

• The ER lumen holds calcium-binding proteins to store calcium ions

• Calcium ATPases (ATP-powered pumps) move calcium ions into the ER, keeping cytoplasmic levels low

• During muscle contraction, calcium ions are released from the sarcoplasmic reticulum

Hydroxylation

• adding OH groups to hydrophobic drugs and toxins

• this increases water solubility for easier excretion

Cytochrome P-450 Enzymes

• known as monooxygenases

• catalyzes hydroxylation reactions

Aryl Hydrocarbon Hydroxylases

• type of cytochrome P-450 enzyme

• breaks down carcinogenic polycyclic hydrocarbons

Sarcoplasmic Reticulum

• specialized type of smooth ER

• found in muscle cells

• regulates calcium ion concentrations

Statins

• a drug that lowers cholesterol by inhibiting enzymes from the smooth ER of liver cells

How does steroid (lipid) biosynthesis work in smooth ER?

• Cells that produce cholesterol and steroid hormones have large amounts of smooth ER

• Key enzymes for cholesterol synthesis are located in the smooth ER of liver cells

• Statins is a drug that lower cholesterol by inhibiting these enzymes

• In plant cells, the smooth ER associates with plastids and helps synthesize phytohormones (plant hormones)

Which of the following statements are true?

1. The rough ER is expected to contain abundant lipids and steroids

2. The smooth ER is expected to contain majority of secreted proteins 

3. The rough ER possess a lot of detoxification enzymes

4. The smooth ER is a major storage compartment of potassium ions

5. The smooth ER is a major storage compartment of calcium ions 

The smooth ER is a major storage compartment of calcium ions 

Membrane Formation by the ER

• Rough and smooth ER both contribute to membrane formation

• The ER is the main source of membrane lipids (phospholipids, cholesterol)

• Fatty acids are made in the cytoplasm and added to the cytosolic side of the ER membrane

• Flippases (phospholipid translocators) move certain lipids to the lumenal side

• This ensures that both sides of the membrane are populated with phospholipids

• This selective transfer contributes to membrane asymmetry, where the lipid composition differs between the two sides of the bilayer

Flippase

• phospholipid translocator that moves certain lipids to the lumenal side from cytosolic side of the ER membrane

• aids in the formation of a membrane by the ER

Golgi Complex

• series of flattened membrane-bounded cisternae

• involves golgi stack (3-8 cisternae)

• some cells have one large cisternae near the nucleus

• other cells have many golgi stacks to handle high volume of lipid and protein processing

• cisternae are organized: cis-Golgi, medial-Golgi and trans-Golgi

• golgi complex is functionally and physically linked to the ER

• lipids and proteins synthesized in ER are transported to golgi for further processing

• central role in membrane and protein trafficking

Transport Vesicles

• both ER and Golgi complex are surrounded by these vesicles

• they carry lipids and proteins from ER to Golgi complex

• the golgi complex lumen is part of the endomembrane system (intracisternal space)

What are the two faces of the golgi stack?

1. cis-Golgi network (CGN): oriented towards the ER

2. trans-Golgi network (TGN): oriented away from ER (opposite)

Golgi Network

• proteins and lipids enter ER and leave the Golgi complex via transport vesicles that bud off the tips of the trans-Golgi

• each region contains specific proteins and enzymes that are unique to its function

Medial Cisternae

• where most protein processing occurs between TGN and CGN

How do lipids and proteins flow through the golgi complex?

1. stationary cisternae model

2. cisternal maturation model

Stationary Cisternae Model

• the golgi cisternae is a fixed structure, does not move

• materials move between cisternae by shuttle vesicles

• these vesicles bud off from one cisternae and fuse with the next

• movement of vesicles occur in cis-to-trans direction

Cisternal Maturation Model

• Cisternae themselves move and change as they progress through the Golgi (cis → trans).

• As they mature, enzymes are sent backwards through vesicles

• this ensures that each region is equipped for its specific function

Why are both cisternae models not mutually exclusive?

These models can occur simultaneously because:

• Both models involve transport vesicles carrying sorted cargo for specific destinations within or outside the Golgi

• Evidence shows both models work together in the Golgi

  • Some materials move via vesicles (stationary model)

  • Others move through maturing cisternae (maturation model)

• Experiments (like tracking cargo and fluorescence microscopy) support:

  • Cisternal maturation over time

  • Presence of vesicle movement between compartments

Anterograde Transport

• Movement of material from the Golgi, towards the plasma membrane

• Secretory granules carry proteins/lipids

• They fuse with the membrane and release contents by exocytosis

• During this process, a small portion of the ER membrane becomes apart of the plasma membrane

• Lipid flow must be balanced to maintain membrane stability

Secretory Granules

• contains proteins and lipids

• involved in anterograde transport (from Golgi to plasma membrane)

• fuse with plasma membrane and release content via exocytosis

Retrograde Transport

• Moves vesicles from the Golgi back to the ER

• Returns some membrane components to balance lipid flow

• Recycles proteins and lipids needed to form new vesicles

A protein you are studying is encoded by a nuclear gene and is constitutively secreted from cells. Therefore, this protein must be transiently found in:

1. Anterograde transition vesicles

2. Retrograde transition vesicles

3. Smooth ER

4. Lysosomes

5. All of the above 

1. Anterograde transition vesicles

   • the protein is being secreted into the cell (movement of material from Golgi to plasma membrane)

How do the ER and Golgi function in protein glycosylation?”

1. N-Linked Glycosylation

   • addition of an oligosaaccharide to the nitrogen atom on the amide group of certain (Asn) residues in the protein

2. O-Linked Glycosylation

• addition of an oligosaccharide to the oxygen atom on the hydroxyl group of Serine (Ser) or Threonine (Thr) residues

N-linked Glycosylation

• addition of an oligosaccharide to the nitrogen atom on an amide group of certain (Asn) residues in the protein

O-linked Glycosylation

• addition of an oligosaccharide to the oxygen atom on an hydroxyl group of Ser or Thr residues

Glycosylation

• adding carbohydrate side chains to proteins, resulting in glycoproteins

• side chains are further processed and modified through enzymatic reactions

• glycosylation occurs in ER and Golgi (protein processing)

How does initial glycosylation occur in the ER?

• Starts on the cytosolic side of the ER; later steps occur in the ER lumen

• A core oligosaccharide (2 N-acetylglucosamine, 9 mannose, 3 glucose) is added to the growing protein

• This process is called N-linked glycosylation, and it happens co-translationally (as the polypeptide is being synthesized)

• The oligosaccharide is then trimmed and modified to support proper protein folding

How does proper folding occur in the ER?

1. Calnexin or Calreticulin:

   • Chaperones like Calnexin (CNX) and Calreticulin (CRT) bind to glycoproteins to stabilize them during folding

   2. ERp57 Thiol Oxidoreductase:

   • ERp57, an enzyme, helps form disulfide bonds

   • Once properly folded, the complex dissociates, the final glucose is removed, the protein is packaged into by vesicles and sent to the Golgi

   • Misfolded proteins are kept in the ER for refolding or sent to ERAD (ER-associated degradation) for disposal

A protein you are studying is normally decorated with oligosaccharides. In mutant cells, the protein is produced but no longer decorated with oligosaccharides. Therefore, in the mutant cells: 

1. The protein is no longer synthesized by the rough ER

2. There must be a problem with processing in the ER or Golgi

3. There must be a problem with processing in the CGN or TGN

4. There must be a problem with processing in the CGN only 

5. There must be a problem with processing in the TGN only

1. There must be a problem with processing in the ER or Golgi

What happens during final glycosylation in the Golgi?

• Glycoproteins move cis → medial → trans Golgi for processing

• Terminal glycosylation creates diversity by:

  • Removing parts of the core oligosaccharide

  • Adding new sugars like GlcNAc, galactose, and others

• Glycosyl transferases are enzymes that add these sugars at specific sites on the glycoprotein

• Final processing prepares proteins for their specific destinations