L8: Membrane trafficking II

Question 1

Biosynthetic vs endocytic route

Answer 1

Biosynthetic (Secretory) Pathway
Starting point: Begins in the endoplasmic reticulum (ER).
Is between the ER and golgi
Protein production:

Secretory and transmembrane proteins are synthesized by ribosomes on the ER membrane.

Proteins are either:

Inserted into the ER membrane, or

Released into the ER lumen.

Cargo transport:

Proteins move from the ER → Golgi apparatus.

In the Golgi, they are modified (e.g., addition of carbohydrates and sugars – post-translational modification).

Final destinations:

Plasma membrane (for secretion),

Storage granules,

Endosomes (early or late).

🔷 Endocytic Pathway
Starting point: Begins at the plasma membrane (PM).

Process:

External materials are internalized into the cell via endocytic vesicles.

Vesicles deliver content to the early endosome.

From early endosome, cargo can go to:

Plasma membrane (recycling),

Golgi apparatus,

Lysosome (for degradation).

Question 2

ER structure

Answer 2

🟩 Endoplasmic Reticulum (ER)
Structure:

Continuous membrane-bound organelle.

Network of interconnected tubules and flattened sacs.

Bilayer structure, connected with the nuclear envelope.

Stretches from the nucleus to the cell periphery.

Forms a high surface area organelle.

Types:

Rough ER (RER):

Covered with ribosomes → site of protein synthesis.

Mostly sheet-like in structure.

Newly made proteins are inserted into the ER membrane or ER lumen.

Smooth ER (SER):

No ribosomes.

Mainly tubular in structure.

Major site for lipid biosynthesis.

Functions:

Synthesis of transmembrane and secretory proteins.

Primary site of lipid synthesis (almost all cellular lipids made here).

Lipids embedded in ER membrane → delivered to other organelles via vesicular or non-vesicular pathways.

Largest organelle in the c

Question 3

golgi structure

Answer 3

🟨 Golgi Apparatus
Structure:

Composed of stacked sheets of flattened membrane sacs called cisternae.

Typically about 8 cisternae visible under electron microscopy.

Ordered with cis, medial, and trans regions.

Ends in the trans-Golgi network (TGN), where vesicles bud off.

Functions:

Modifies, packages, and sorts secretory cargo (soluble and membrane-bound proteins).

Main site for glycosylation (addition of sugar groups).

Receives cargo from the ER at the cis face.

Sends cargo from the trans face to the plasma membrane or other destinations.

Position in cell:

Positioned near the microtubule organizing center (MTOC) at the cell center.

Acts as a hub for microtubule-based transport:

Receives cargo via microtubules.

Sends out cargo along the same tracks.

Question 4

forward transport

Answer 4

ER to the Golgi
🟧 Forward (ER to Golgi) Transport – Vesicular Trafficking
General Process:

Vesicles bud from one compartment (e.g. ER) and fuse with the next (e.g. Golgi).

Transport involves vesicle coats that help form the vesicle and select cargo.

🟣 Types of Vesicle Coats:
🔹 COPII:
Found on ER membranes.

Drives anterograde (forward) transport:

From ER → Golgi.

Moves newly synthesized proteins from ER to Golgi for further modification.

🔹 COPI:
Found on Golgi membranes.

Drives retrograde (backward) transport:

From Golgi → ER.

Between Golgi cisternae (late → early Golgi elements).

Used for:

Retrieving ER-resident proteins that escaped accidentally.

Recycling vesicle components.

🔸 Key Differences from Endocytic Pathway:
Endocytic pathway mainly uses clathrin-coated vesicles.

Secretory pathway (ER↔Golgi) uses COPI and COPII, not clathrin.

Question 5

cargo selection at ER exit sites (ERES) and COP-II budding

Answer 5

argo Selection and COPII Budding at ER Exit Sites (ERES)
🧩 Overview:
ER Exit Sites (ERES): Specialized regions on the ER where COPII-coated vesicles bud off.

COPII vesicles carry cargo (proteins) from the ER to the Golgi apparatus.

🔹 Key Players & Steps:
1. Sar1 Activation (Initiation of Vesicle Formation):
Sar1: A small GTPase located at the ER membrane.

Sar1-GEF (Guanine Nucleotide Exchange Factor) activates Sar1 by exchanging GDP for GTP.

Activated Sar1-GTP embeds an amphipathic α-helix into the ER membrane.

This creates a Sar1-positive domain, marking the ER exit site.

2. COPII Coat Assembly:
   🔸 Inner Coat – Cargo Selection:
   Sar1-GTP recruits the Sec23/Sec24 complex.

Sec24: Binds to cargo proteins (usually transmembrane or cargo receptors).

Sec23: Acts as a GTPase-activating protein (GAP) for Sar1.

🔸 Outer Coat – Vesicle Formation:
Sec13/Sec31 is recruited next to complete the outer COPII coat.

This complex deforms the ER membrane, promoting vesicle budding.

🧪 Constituents of COPII Coat:
Inner layer: Sar1, Sec23, Sec24.

Outer layer: Sec13, Sec31.

📦 Function:
The full COPII coat enables cargo sorting, vesicle formation, and departure from the ER.

This forms a transport intermediate that delivers proteins to the Golgi.

Question 6

cargo sorting at ERES

Answer 6

Transmembrane cargo (gen in er and needs to go to diff organelle) engages
COP-II coat
* Soluble cargo binds export
receptors
* Chaperones sequester cargo
until it is properly folded (e.g.,
cover ER exit signals)
* Excess unfolded cargo triggers
the UPR – the unfolded protein
respsonse

Export receptors in the ER membrane bind to soluble cargo in the lumen.

Chaperones in the ER help fold proteins and ensure only properly folded proteins are exported.

🔹 Key Steps in Cargo Selection:
1. Chaperones & Protein Folding:
Many chaperones assist in protein folding within the ER lumen.

These chaperones bind to misfolded or unfolded proteins to prevent them from exiting the ER.

Properly folded soluble proteins expose specific exit signals that need to be recognized for export.

2. Chaperone Binding to Exit Signals:
   Chaperones often mask the exit signals on proteins until they are properly folded.

Unfolded or misfolded proteins are retained in the ER by chaperones.

3. Release of Soluble Proteins:
   Once a protein is properly folded, the chaperone releases the protein.

The protein’s exit signal can now bind to export receptors located in the ER membrane.

4. Cargo Incorporation into COPII Vesicles:
   The export receptor, now bound to the protein with the exit signal, is incorporated into the COPII vesicle.

This ensures only properly folded soluble proteins are transported in COPII vesicles from the ER to the Golgi.

🔸 Function of Chaperones in Cargo Selection:
Chaperones act as quality control agents, preventing misfolded proteins from leaving the ER.

They concentrate properly folded proteins in COPII vesicles, ensuring only functional cargo is exported.

Question 7

retrograde transport

Answer 7

Retrograde transport
Acts as a quality control mechanism to make sure any proteins that should stay in er lumen like the chaperones dont leave by accident and go back.
Uses cop-1
Cop-1 uses KDEL receptor to select cargo that should not be in golgi, should be in er.
KDEL receptor binds KDEL
displaying cargo at acidic pH
* KDEL receptor releases KDEL
displaying cargo at neutral pH
* KDEL binding to KDEL receptor
exposes the COP-I binding ER-
retrieval motif
* Allows KDEL receptor to pick up
cargo in the Golgi (acidic) and
release it in the ER (neutral)

KDEL sequence: A specific series of amino acid residues (Lys-Asp-Glu-Leu) found in ER-resident proteins and chaperones.

KDEL receptor: Located in the Golgi, binds KDEL-containing cargo and directs it back to the ER via COP-I vesicles.

🔹 Key Concepts:
1. KDEL Sequence and Binding:
KDEL is a sorting signal found on ER-resident soluble proteins (such as chaperones).

In the Golgi, the pH is more acidic compared to the ER.

At acidic pH (in the Golgi), the KDEL receptor binds KDEL-containing cargo.

2. COP-I Vesicle Formation:
   The KDEL receptor binds the cargo and incorporates it into COP-I-coated vesicles.

COP-I vesicles mediate the retrograde trafficking of ER-resident proteins back to the ER.

3. pH-Sensitive Binding:
   In the Golgi, the acidic environment facilitates strong binding between the KDEL receptor and the cargo.

As the vesicle moves toward the ER, the pH becomes less acidic (more neutral).

At this neutral pH, the KDEL receptor releases its cargo in the ER, as the binding is weaker at a higher pH.

🔸 Function of the KDEL Receptor:
KDEL receptor ensures the recycling of ER-resident proteins by recognizing and binding them in the Golgi.

It facilitates the retrograde transport of these proteins back to the ER via COP-I vesicles.

Question 8

how does cargo move through the golgi?

Answer 8

2 models of transport from cis cisternae, medial cisternae then to trans cisternae.
Cisternal Maturation Model
Cargo Delivery: Cargo is initially delivered to the cis cisterna of the Golgi.

Maturation Process:

As cargo moves through the Golgi, COP-I-dependent budding occurs from one cisterna to another.

This maturation process allows the cis cisternae to gradually mature into trans cisternae.

Progression Through the Golgi: The cisternae themselves mature as they move through the Golgi, from cis to trans.

The Golgi cisternae mature by acquiring new enzymes and losing unnecessary cargo through COP-I budding.

2. Vesicle Transport Model
   Cisternae as Static Structures: In this model, the cisternae themselves are static and do not move.

Vesicular Budding: Instead of cisternae maturing, individual vesicles bud off from one cisterna and deliver their cargo to the next cisterna in the Golgi stack.

COP-I-coated vesicles move in the opposite direction, retrieving cargo or enzymes from later cisternae for the earlier ones.

Cargo Movement: This model suggests that cargo is transported by vesicles rather than by maturation of the cisternae themselves.

🔹 Comparison:
Cisternal Maturation Model: Cisternae mature as they move through the Golgi stack, with cargo being processed and modified.

Vesicle Transport Model: Cisternae remain static, and vesicles transport cargo between cisternae.

Question 9

what happens to the secretory cargo in the golgi?

Answer 9

Sequential modification of
carbohydrates on secreted
proteins
* Allows proper folding of
secreted cargo
* Protects secreted proteins in the
extracellular environment
* Creates sorting signals for
transport to endosomes

Glycosylation and Protein Modifications at the Plasma Membrane and Golgi
1. Glycosylation of Plasma Membrane Proteins:
Heavily Glycosylated Proteins: Many proteins on the outer face of the plasma membrane are heavily glycosylated, meaning they have many sugar modifications attached.

Functions of Carbohydrates:

Protection: The carbohydrates help protect the proteins from the external environment.

Signaling Molecules: These sugar modifications can act as signaling molecules, such as sorting signals, which help the cell recognize or sort molecules.

Lectins: Lectins, proteins that bind to carbohydrates, are often modified by these sugars, which alters their cell surface properties.

2. Secretory Proteins and Glycosylation:
   Many secretory proteins are also modified by carbohydrates as they move through the cell.

🟩 Cargo Movement and Post-Translational Modifications in the Golgi:
1. Cargo Arrival at the Golgi:
Cargo from the ER arrives at the cis Golgi network (CGN), which is the entry point for proteins destined for modification.

2. Post-Translational Modifications in the Golgi:
   Once in the Golgi, proteins undergo a series of post-translational modifications, primarily glycosylation.

Sequential Modifications:

Each cisterna in the Golgi stack contains specific enzymes that catalyze different modifications.

Sequential cisternae in the Golgi allow for step-by-step modification of proteins, often by adding different carbohydrates (such as mannose, glucose, or sialic acid).

🔹 Summary of Key Concepts:
Carbohydrate Modifications:

Protect proteins from the environment.

Act as signaling molecules or sorting signals (e.g., lectins).

Golgi Modifications:

Cargo from the ER enters at the cis Golgi network and undergoes sequential glycosylation.

Each cisterna in the Golgi stack has enzymes for specific post-translational modifications.

Question 10

what happens to the cargo after golgi?

Answer 10

3 major destinations for cargo leaving the TGN:
* Endosomal network
* Plasma membrane (constitutive)
* Secretory granules (regulatory)
TGN Overview:
The Trans Golgi Network (TGN) is a dynamic organelle involved in sorting, sequestering, and packaging cargo.

After post-translational modifications in the Golgi, cargo is delivered to the TGN, where it is sorted and sent to different destinations within the cell.

🟩 3 Main Pathways for Cargo from TGN:
1. Constitutive Secretory Pathway:
Cargo is transported directly from the TGN to the plasma membrane (PM) or outside the cell.

This is a continuous, unregulated process where proteins and lipids are constantly secreted to maintain cell functions.

2. Regulated Secretory Pathway:
   Cargo is packaged in secretory granules at the TGN and stored until a stimulus triggers its release.

Matured granules fuse with the PM and release their content upon signaling (e.g., hormones or neurotransmitters).

3. Endocytic Pathway:
   Cargo can be directed to the endocytic network for further processing.

Provides materials needed for the endocytic and lysosomal network to function, such as proteins involved in recycling or degradation.

🟩 SNARE-Mediated Fusion:
SNARE proteins are involved in the fusion of vesicles with their target membranes.

This is crucial for cargo delivery in all three pathways (Constitutive, Regulated, and Endocytic).

SNAREs help to ensure the specificity of membrane fusion and cargo delivery to the correct destination.

🔹 Summary of Key Concepts:
The TGN serves as a sorting station for cargo after it’s modified in the Golgi.

Three primary routes for cargo:

Constitutive Secretory Pathway: Constant release to the PM or outside.

Regulated Secretory Pathway: Stored in secretory granules, released upon stimulation.

Endocytic Pathway: Delivered to the endocytic network for further processing or degradation.

SNARE proteins mediate the fusion of vesicles with their target membranes in each of these pathways.