L8: Membrane trafficking II part 2 Flashcards
(10 cards)
constitutive vs regulated secretion
Constitutive Secretion
Purpose: Delivers new lipids and proteins to the plasma membrane (PM). This supports general turnover and maintains cell function.
Key Features:
Unregulated: It happens continuously, regardless of external signals.
Vesicle Formation: Vesicles bud off from the trans-Golgi network (TGN) and fuse with the PM.
Cargo: New receptors, lipids, and proteins are delivered to the PM or secreted to the outside world.
Function:
Replenish surface content of the PM, ensuring that it stays functional and maintains cellular integrity.
🟩 Regulated Secretion
Purpose: Stores specific secretory proteins in vesicles until they are needed for cell signaling.
Key Features:
Signal-Mediated: It is triggered by external signals (e.g., hormones, neurotransmitters, cytokines).
Vesicle Formation: Secretory vesicles bud from the TGN, but instead of fusing with the PM immediately, they remain static in the cytoplasm.
Release upon Stimulus: When an extracellular signal activates a signaling cascade, the vesicle fuses with the PM and releases its cargo.
Examples of Secretory Proteins:
Insulin
Neurotransmitters
Cytokines
Perforin
Granzymes
Function:
Allows cells to produce changes in nearby cells in response to stimuli.
maturation of secretory granules: cargo concentration
Acidification and Cargo Concentration in Secretory Vesicles
H⁺ ATPase in Secretory Vesicle Membranes
A proton pump (H⁺ ATPase) in the vesicle membrane helps acidify the contents of the vesicle.
This acidification leads to a phase change in the proteins within the vesicle, causing them to condense and hyperconcentrate.
Clathrin-Mediated Budding
Clathrin helps remove excess membrane and fluid from the immature secretory vesicle, further concentrating the cargo inside.
This fluid removal helps make the contents of the vesicle as dense as possible.
Selective Aggregation of Cargo
pH changes and increased concentration of the vesicle contents lead to the selective aggregation of proteins.
Proteins of the same class tend to clump together, forming aggregates that are more concentrated and stable in the vesicle.
🟩 Immature vs. Mature Secretory Granules
Immature Secretory Vesicles:
Initially formed from the Golgi network.
Cargo is packaged and concentrated, but not fully matured.
Mature Secretory Granules:
Once cargo concentration is achieved via acidification and clathrin-mediated budding, the granule is considered mature.
These vesicles can stay in the cytoplasm for a long time until triggered by external signals.
🟩 Examples of Specialized Secretory Cells
Mast Cells: Secrete histamine in response to immune signals (important for allergic reactions).
Goblet Cells: Secrete mucus to protect and lubricate surfaces, especially in the respiratory and gastrointestinal systems.
Neurons: Release neurotransmitters (NTs) to communicate with other neurons or muscles.
Endocrine/Exocrine Cells:
Endocrine cells secrete hormones into the bloodstream.
Exocrine cells secrete substances like digestive enzymes into ducts or body cavities.
the endocytic pathway
The Endocytic Pathway
The endocytic pathway is responsible for internalizing material that cells need to process or degrade. It involves the following steps:
Materials are internalized from the plasma membrane (PM).
The internalized cargo travels through early endosomes, late endosomes, and ultimately fuses with lysosomes, where it is processed and degraded.
Lysosome:
The lysosome is a terminal degradative organelle of the cell.
It contains proteases and lipases that break down proteins and lipids.
Acidic environment: The lysosome has a very low pH, maintained by H⁺ ATPases that pump protons into the lumen.
It serves to degrade proteins, recycle amino acids and lipids, and generate building blocks for biosynthesis.
🟩 Degradation of Proteins
Proteins are degraded by different pathways depending on whether they are soluble or membrane-bound.
- Proteasomal Degradation (Soluble Proteins)
Proteasome: A complex that degrades soluble proteins in the cytoplasm.
Proteins are tagged with ubiquitin (a small protein) to mark them for degradation.
Ubiquitinated proteins are recognized by the proteasome, where they are unfolded and broken down into smaller peptides.
Proteasomal degradation is essential for regulating the cell’s protein homeostasis (proteostasis).
- Lysosomal Degradation (Membrane Proteins)
Transmembrane proteins (TM proteins):
TM proteins cannot be degraded directly by the proteasome because they are embedded in the membrane.
They are first ubiquitinated and then sorted into endosomes.
The endosomes mature into late endosomes, which then fuse with lysosomes for degradation.
Lysosomes provide the only site for the degradation of membrane proteins.
Proteolytic and hydrolytic enzymes in the lysosome break down the TM proteins into smaller components.
🟩 Lysosomal Features
Proton pump (H⁺ ATPase): Ensures an acidic environment within the lysosome by pumping protons into its lumen using ATP hydrolysis.
Proteolytic and Hydrolytic: The lysosome contains enzymes that catalyze the breakdown of proteins and lipids.
Free amino acids: After degradation, free amino acids are transported out of the lysosome for recycling and biosynthesis.
entering the endosomal system
Entering the endosomal system.
3 ways to enter:
Uptake of proteins from the cell surface
* Phagocytosis: uptake of large particles
(debris/other cells/particles) by large PM folds
* Pinocytosis: non-specific uptake of fluid phase
by smaller plasma membrane folds
* Receptor mediated endocytosis: selective,
signal mediated uptake of cargo
receptor mediated endocytosis
Receptor-Mediated Endocytosis
Receptor-mediated endocytosis is a highly specific process by which cells internalize cargo from the extracellular space using specialized proteins and membrane modifications.
Cargo Sequestration at the Plasma Membrane (PM):
Cargo: Proteins, lipids, or other molecules are concentrated at specific regions of the plasma membrane (PM).
This cargo is typically recognized by cell surface receptors that are selectively concentrated at these sites.
Invagination and Membrane Shaping:
Invaginations: The membrane begins to curve inward at these regions, forming pockets or invaginations that concentrate the cargo.
Bar proteins: Proteins with bar domains (e.g., Amphiphysin, Endophilin) help to shape and bend the membrane. These proteins induce the necessary curvature in the membrane, driving invagination and promoting the formation of a vesicle.
Vesicle Formation and Scission:
As the invagination deepens, the membrane continues to bend, eventually pinching off to form a transport vesicle.
Scission: The final step involves the severing of the vesicle from the plasma membrane, a process facilitated by dynamin, a GTPase that wraps around the neck of the vesicle and helps to snip it off from the membrane.
Cargo Transport: Once the vesicle is internalized, it can be transported through the cell to endosomal compartments for processing, recycling, or degradation, depending on the type of cargo.
🟩 Receptor-Mediated Endocytosis Steps
Receptor Binding: Specific receptors on the plasma membrane recognize and bind the cargo (e.g., low-density lipoprotein (LDL) particles bound to the LDL receptor).
Invagination: The plasma membrane at the receptor site begins to curve inward due to the action of bar proteins.
Vesicle Formation: The membrane continues to curve, and dynamin assists in pinching off the vesicle.
Endocytosis: The vesicle is fully formed and internalized into the cell for processing.
Cargo Sorting: The vesicle fuses with early endosomes, where the cargo is either processed or sent to the lysosome for degradation.
clathrin-mediated endocytosis
Clathrin-mediated endocytosis is a key process for internalizing cargo at the plasma membrane (PM), primarily used for the internalization of receptors and their bound ligands. Here’s a breakdown of the process:
Clathrin and Triskelion Formation:
Clathrin: A protein that assembles into a triskelion structure (three-legged structure) and forms a mesh-like coat around the membrane.
Triskelions nest together to form a spherical or basket-like shape, creating a clathrin-coated pit that buds from the plasma membrane.
Cargo Concentration:
The clathrin coat assembles at specific regions of the PM, where it not only forms a structural scaffold but also concentrates cargo by recruiting cargo receptors on the cell surface. This allows for efficient internalization of materials.
Dynamin and Vesicle Scission:
Dynamin: A GTPase that plays a crucial role in the scission of the vesicle from the plasma membrane. Dynamin assembles at the neck of the budding vesicle and uses energy from GTP hydrolysis to tighten the neck, ultimately severing the vesicle from the membrane.
Clathrin-Coated Vesicle Formation:
After the vesicle is pinched off from the plasma membrane, it remains covered by the clathrin coat.
The clathrin disassembles after vesicle formation, leaving behind a free vesicle that can then fuse with early endosomes or other target organelles.
Fusion with Target Organelle:
The free vesicle fuses with its target organelle (e.g., early endosomes) for processing, cargo sorting, or degradation.
🟩 Key Proteins in Clathrin-Mediated Endocytosis
Clathrin: Forms the triskelion structure that assembles into a coat around the vesicle.
Dynamin: GTPase responsible for scission and pinching off the vesicle from the membrane.
Cargo Receptors: Bind and concentrate cargo at the plasma membrane for internalization.
Adaptor Proteins (e.g., AP2): Bind clathrin and the cargo receptors, aiding the assembly of the clathrin coat and vesicle formation.
evolutionary similarity of coat proteins
The coat proteins clathrin, COP-I, and COP-II share evolutionary similarities in their structural organization and function in vesicular trafficking. While each coat protein is specific to distinct transport routes, they all function similarly in shaping and budding vesicles through membrane deformation. Here’s how these proteins are similar:
Common Feature: Scaffold Formation
Scaffolding proteins: Clathrin, COP-I, and COP-II proteins all form scaffold-like structures that deform the underlying membrane and induce vesicle budding.
These scaffolds help concentrate cargo, shape the vesicle, and provide a framework for other proteins (e.g., dynamin, SNAREs) involved in membrane fission and fusion.
Membrane Deformation and Vesicle Formation:
The structural units of these proteins, whether they are triskelions in clathrin or COP-I and COP-II subunits, interact with the membrane to generate the mechanical force required to bend and pinch off vesicles.
🟩 Clathrin Structure
Clathrin forms a 3-lobed structure known as a triskelion.
Each triskelion consists of three heavy chains and three light chains that come together to form a three-dimensional cage-like structure around the vesicle.
The triskelions assemble to form a meshwork, providing structural support for the budding vesicle.
Clathrin is primarily involved in clathrin-mediated endocytosis (e.g., receptor internalization).
🟩 COP-II Structure
COP-II forms a coat that is mainly involved in anterograde transport (from ER to Golgi).
The coat consists of a complex of proteins, including Sar1, Sec23, Sec24, Sec13, and Sec31.
Sar1 initiates the process by embedding into the membrane and recruiting other COP-II subunits. These subunits assemble into a lattice-like structure that drives vesicle formation at the ER exit sites.
COP-II vesicles are used for exporting newly synthesized proteins from the ER to the Golgi apparatus.
🟩 COP-I Structure
COP-I primarily functions in retrograde transport (from Golgi to ER and between Golgi cisternae).
COP-I is composed of seven subunits: α, β, β’, γ, δ, ζ, and the ARF GTPase.
The structure of COP-I is similar to COP-II in that it forms lattice-like scaffolds. These subunits assemble on Golgi membranes and help generate vesicles for transporting materials back to the ER or within the Golgi.
COP-I helps in the retrieval of resident ER proteins that have escaped or are required for maintenance of Golgi function.
🟩 Commonalities in Coat Protein Structures
Membrane Bending: All three coats—clathrin, COP-I, and COP-II—generate curvature on the membrane and are involved in vesicle scission. They create tension on the membrane, forcing it to bend and eventually pinch off to form transport vesicles.
Assembly into Lattices: The subunits of clathrin, COP-I, and COP-II form lattice-like structures that impose structural rigidity and force, driving vesicle formation.
Evolutionary Similarity: Although each protein complex serves distinct trafficking pathways, the general mechanism of action—forming a scaffold that bends and shapes the membrane to form vesicles—is a conserved feature across all three families.
molecular structure of coats
Molecular Structure of Different Coats
Clathrin:
Triskelion structure made of three heavy chains and three light chains.
Forms a hexagonal network that wraps around the membrane to generate vesicle shape.
COP-II:
Composed of Sar1, Sec23, Sec24, Sec13, and Sec31.
Sar1 GTPase initiates vesicle budding by embedding into the membrane and recruiting other COP-II components.
cop II (Vesicle) or cop II (tubule structure) for largecargo
COP-I:
Seven subunits (including ARF GTPase and coatomer proteins) form a scaffold that coats the vesicle and promotes its budding.
how is cargo incoporated into forming vesicles
- Adaptor proteins link cargo to forming clathrin-
coated vesicles - Adapter proteins recognize short sequence
motifs in receptor C-termini
Cargo doesn’t directly interact with clathrin. Adaptor proteins link clathrin with cargo: one part binds clathrin triskelia, the other binds cargo. They also bind phosphoinositol lipids like PIP2, concentrating at sites in the membrane to recruit both cargo and clathrin for vesicle formation.
Sequence motifs:
NPXY in LDL receptor
YXXØ in Transferrin receptor
scission of the vesicles
Scission of vesicles: the Dynamit GTPase
* A large GTPase that binds to
necks of budding vesicles
Lost in a fly mutant (shibire)
that displays neuromuscular
defects due to problems in
synaptic vesicle generation
Constricts and separates
vesicle from membrane of
Formation
In the mutants: vesicles form, some protein density around the neck but vesicles can’t bud off. Think dynamin – a mechanical GTPase. Constricts and severs the vesicle membrane from the donor membrane. Uses GTP hydrolysis to power this scission and release the vesicle into the cytoplasm.
