Lecture 12 Flashcards
two secretion pathways responsible for transporting ‘cargo’ from TGN to pm and extracellular space
Constitutive secretion pathway
- Materials are continually transported from the TGN to the plasma membrane via secretory vesicles, following what is considered the ‘default’ pathway.
- This pathway is utilized for proteins that are not selectively sorted through the biosynthetic pathway (i.e., targeted to late endosomes and lysosomes) or by regulated secretion mechanisms.
- These secretory vesicles fuse with the PM through the action of Rabs and SNAREs, releasing their lumenal soluble cargo outside of the cell via exocytosis.
- This cargo typically includes soluble enzymes or materials required for extracellular matrix or cell wall synthesis. Additionally, the vesicle membrane components, which constitute cargo, are incorporated into the plasma membrane, thereby providing new membrane proteins (e.g., cell surface receptors) and lipids to the PM.
two secretion pathways responsible for transporting ‘cargo’ from TGN to pm and extracellular space
Regulated secretion pathway
- involves the packaging of materials at the trans-Golgi network (TGN) into secretory granules. These granules are stored in the cytoplasm and, in response to cellular signals, are targeted to and fuse with the plasma membrane via the action of Rabs and SNAREs, releasing their lumenal cargo outside of the cell through exocytosis.
- Examples of regulated secretion include the release of hormones by endocrine cells and neurotransmitters by nerve cells.
- The process of secretory granule (and secretory vesicle) formation at the TGN, including the involvement of protein coats, is not well understood.
Endocytic pathway…
- operates in opposite direction of secretory pathways
- materials (macromolecules) move into cell via vesiculation of pm and either recycled back to pm or sorted to different intracellular destinations
Two main processes for internalization
- Phagocytosis
- Endocytosis
Phagocytosis
uptake of large, particulate materials from extracellular space by specialized cells
Phagocytosis
Recognition and removal of bacteria by leukocytes
- bacteria identified by immune system as ‘foreign’ material and generates antibodies against bacterial cell-surface components. antibody Fab domain binds bacterial cell-surface protein/sugar(s) process referred to as opsonization
- leukocyte plasma membrane Fc receptors recognize exposed Fc domain on antibodies bound to bacterium
* Fc receptors ‘signal’ re-assembly of actin microfilament network alterations in cytoskeleton result in changes in shape of of leukocyte – cell extensions = pseudopods - leukocyte pm engulfs (pseudopods) bacterium and fuse to form phagosome
- phagosome fuses with lysosome
* phagosome-lysosome fusion blocked by Mycobacterium tuberculosis
* bacteria ‘hijacks’ phagocytic machinery to infect leukocyte (preferred host cell)
* bacterium digested and nutrients released into cytoplasm of leukocyte
Endocytosis encompasses various forms with distinct mechanisms:
- Bulk-phase endocytosis (pinocytosis or ‘cellular drinking’)
- Receptor-mediated endocytosis
Bulk-phase endocytosis (pinocytosis or ‘cellular drinking’) involves..
This involves the non-specific uptake of extracellular fluids, along with plasma membrane proteins and lipids, into small vesicles. The plasma membrane recycles approximately every 20-90 minutes in this process.
Receptor-mediated endocytosis involves
This process involves specific cell-surface receptors on the plasma membrane binding to extracellular ligands. Subsequently, the receptor-ligand complexes are concentrated and internalized within clathrin-coated transport vesicles. This mechanism allows for the selective uptake of particular molecules into the cell.
Examples of materials (macromolecules) internalized by receptor-mediated endocytosis…
• M6P receptor-bound, lysosomal proteins ‘escaped’ from TGN via secretory pathway [Lecture 11]
• Receptor complexes with bound cell signaling hormones (e.g., insulin) or growth factors (e.g., EGF)
• Iron (Fe3+)-bound carrier protein ferrotransferrin recognized by transferrin receptor
• Cholesterol-containing, low-density lipoprotein (LDL) particle recognized by LDL receptor (Fig. 14-25)
Steps of Receptor mediated endocytosis
Step 1
* transmembrane receptor at plasma membrane ‘activated’ by binding to specific extracellular ligand
* cytoplasmic-facing domain of receptor binds to AP2 adaptor ‘coat’ protein – cytoplasmic protein serves as ‘linker’ during clathrin-coat vesicle assembly at pm AP2 ≈ AP1/GGA adaptors during clathrin-coated vesicle assembly at TGN
- receptor-ligand-AP2 complex accumulates in clathrin-coated pit
- specialized regions (indentations) of pm where receptor-ligand complexes are concentrated and endocytic vesicles eventually form
- inner (cytoplasmic) leaflet of pm at coated pit enriched in unique membrane phospholipids membrane lipid ‘microdomain’ – enriched in phosphatidylinositol (PI) (4,5) P2 – serves as signal for recruiting AP2 with bound receptor-ligand into coated pit
- AP2 has multiple binding domains:
- i) PI(4,5)P2,
- ii) cytoplasmic-domains of pm transmembrane receptors (w/ extracellular-bound ‘cargo’), and iii) clathrin
- similar to AP1/GGA adaptors at TGN, AP2 at cytoplasmic face of coated pit forms inner layer of ‘coat’
- AP2 also recruits clathrin triskelions from cytoplasm
- clathrin triskelions self-assemble to form outer scaffolding (cage-like ‘lattice’) of coat on growing endocytic vesicle bud
- clathrin hexagon-to-pentagon (‘lattice’) formation acts as a mechanical driving force for inward curvature of plasma membrane
Step 2
* clathrin-coated vesicle bud ‘pinches off’ from pm via dynamin (and GTP hydrolysis)
* soon after budding, the clathrin coat disassembles from vesicle
* AP2 and clathrin triskelions released into cytoplasm and ‘recycled’ for additional rounds of clathrin-coat endocytic assembly at pm
* nascent, uncoated endocytic vesicle referred to as early endosome
Step 3
* early-late endosome trafficking/docking/fusion mediated by organelle-specific Rabs/Rab effectors & v/t-SNARES
* acidic interior (~pH 5.0-5.5) of late endosome lumen (endocytic vesicle & ECM [pH ~7]) causes dissociation of receptor-ligand complexes
Step 4
* late endosome with ‘free’ soluble ligands fuse with lysosome
* trafficking/docking/fusion mediated by specific Rab/Rab effector and SNARES
Step 5
* ‘free’ receptors recycled back to cell surface (via transport vesicles [type?]) for additional rounds of receptor-mediated endocytosis
* alternatively, membrane-bound receptors degraded within lysosomes
Delivery of membrane proteins to lysosome interior for degradation
• during receptor-mediated endocytosis, ____ recycled back to cell surface OR ____
during receptor-mediated endocytosis, ‘free’ receptors recycled back to cell surface OR delivered to lysosome interior for degradation
How are membrane proteins delivered to interior (lumen) of lysosome for degradation?
degradation of endocytosed membrane proteins (e.g., cell-surface receptors) involves inward budding of vesicles into late endosome interior forming a multivesicular late endosome or multivesicular body (MVB)
How are membrane proteins delivered to interior (lumen) of lysosome for degradation?
• Step 1: involves sorting at MVB of nascent resident lysosomal membrane proteins from TGN (via biosynthetic pathway) from membrane proteins destined (via endocytic pathway step 2 for degradation in lysosomes
• MVB has unique morphology - contains numerous intralumenal vesicles similar in size to transport vesicles, but opposite topology: MVB vesicles bud away from the cytoplasm (unlike COPI/II and clathrin vesicles - bud towards cytoplasm)
• Step 3: MVB vesicles selectively contain membrane proteins destined for degradation in lysosome interior
What is ESCRT
Endosomal Sorting Complex Required for Transport
multi-protein complex – soluble (cytosolic) protein constituents recruited to MVB surface - mediate membrane ‘cargo’ protein selection and inward vesicle budding
