Endomembrane System Part 5 Flashcards
What are the secretion pathways responsible for?
Responsible for transporting ‘cargo’ from TGN to pm and extracellular space
What is the Constitutive Secretion Pathway?
Materials are continually transported (via secretory vesicles) from TGN to pm
Vesicles fuse (via Rabs and SNAREs) with the pm and release (exocytosis) their luminal (soluble) ‘cargo’ outside of the cell
Vesicle membrane ‘cargo’ components are incorporated into the plasma membrane
Provides new membrane proteins (e.g., cell surface receptors) and lipids to pm
Considered the default pathway
Pathway for proteins not selectively sorted through the biosynthetic pathway
i.e. targeted to late endosomes and lysosomes OR by regulated secretion
What is the Regulated Secretion Pathway?
Materials at TGN packaged into secretory granules
In response to a cellular signal
Granules stored in the cytoplasm target to and fuse with (via Rabs and SNAREs) plasma membrane and release (exocytosis) their luminal ‘cargo’ outside of the cell
e.g., the release of hormones by endocrine cells
Secretory granule (& secretory vesicle) formation at TGN (including protein (coat(s)) is not well understood
What is the endocytic pathway?
Operates in the opposite direction of secretory pathways
Materials (macromolecules) move into the cell via vesiculation of plasma membrane and either recycled back to pm or sorted to different intracellular destinations
Different fates in cell depend on material internalized
Two main processes for internalization:
—-> Phagocytosis- uptake of large, particulate materials from extracellular space by specialized cells, such as white blood cells (leukocytes)
—> Endocytosis – different forms with varied mechanisms
What is phagocytosis?
Recognition and removal of bacteria by leukocytes
Bacteria identified as immune system as ‘foreign’ material and generates antibodies against bacterial cell surface components
1) Antibody Fab domain binds bacterial cell-surface protein/sugar(s) – referred to as opsonization
2) Leukocyte plasma membrane Fc receptors recognize exposed Fc domain on antibodies bound to the bacterium
Fc receptors ‘signal’ re-assembly of actin microfilament network
Alterations in cytoskeleton result in changes in the shape of leukocyte
3) Leukocyte pm engulfs (pseudopods) bacterium and fuse to form a phagosome
4) Phagosome fuses with a lysosome
Phagosome-lysosome fusion blocked by Mycobacterium tuberculosis
Bacteria ‘hijacks’ phagocytic machinery to infect leukocyte (preferred host cell)
Bacterium digested and nutrients released into the cytoplasm of leukocyte
What is endocytosis?
Bulk phase endocytosis (pinocytosis or ‘cellular drinking’)
Non-specific uptake of extracellular fluids and plasma membrane proteins and lipids into small vesicles
pm recycles ever approx. 20-90 min
Receptor-mediated endocytosis
Specific cell-surface (pm) receptor binds extracellular ligand(s) and receptor-ligand complexes subsequently concentrated and internalized in clathrin-coated transport vesicles
Examples of materials (macromolecules) internalized by receptor-mediated endocytosis:
M6P receptor-bound, lysosomal proteins ‘escaped’ from TGN via the secretory pathway
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
What are the steps of receptor-mediated endocytosis?
1) Transmembrane receptor at plasma membrane becomes ‘activated’ by binding to a specific extracellular ligand
The extracellular-facing domain of pm LDL receptor binds to LDL particle (‘cargo’)
The cytoplasmic-facing domain of receptor binds to AP2 adaptor ‘coat’ protein
Cytoplasmic protein serves as ‘linker’ during clathrin-coat vesicle assembly (= AP1/GGA adaptors during clathrin-coated vesicle assembly at TGN)
1) Receptor-ligand-AP2-complex accumulates in clathrin-coated pit
Where clathrin is involved in forming vesicle that will eventually if bud off the coated pit
Specialized regions (indentations) of pm where receptor-ligand complexes are concentrated, and endocytic vesicles eventually formed
Inner leaflet of pm at coated pit enriched in unique membrane phospholipids
Lipid ‘microdomain’ – enriched in phosphatidylinositol (PI)(4,5)P2 – serves as a signal for recruiting AP2 with bound receptor-ligand into the coated pit
AP2 has multiple binding domains: PI(4,5)P2, cytoplasmic domains of pm transmembrane receptors with extracellular-bound ‘cargo’ and clathrin
Similar to AP1/GGA adaptors at TGN, AP2 at the cytoplasmic face of coated pit forms the inner layer of ‘coat’
1) AP2 recruits clathrin triskelions from the cytoplasm
Clathrin triskelions self-assemble to form outer scaffolding (cage-like ‘lattice’) of coat on growing endocytic vesicle bud
Clathrin hexagon-to-polygon (‘lattice’) formation acts as a mechanical driving force for inward curvature of plasma membrane
2) Clathrin-coated vesicle bud ‘pinches off’ from pia via dynamin and GTP hydrolysis
Soon after budding, the clathrin coat disassembles from the vesicle
AP2 and clathrin triskelions released into the cytoplasm and ‘recycled’ for additional rounds of clathrin-coat endocytic assembly of pm
Nascent, uncoated endocytic vesicle referred to the as early endosome
3) Early endosome fuses with the late endosome
Early endosome trafficking/docking/fusion with late endosome mediated by organelle-specific Rabs/Rab effectors and v/t-SNAREs
Acidic interior (pH 5.0-5.5) of late endosome lumen (endocytic vesicle(pH = 7)) causes dissociation of receptor-ligand complexes
4) Late endosome with ‘free’ soluble ligands fuse with the lysosome
trafficking/docking/fusion mediated by specific Rabs/Rab effectors and SNAREs
5) ‘Free’ receptors recycled back to the cell surface (via transport vesicles) for additional rounds of receptor-mediated endocytosis
Alternatively, membrane-bound receptors degraded within lysosomes
In the lysosome, Ligand(s) degraded by lysosomal acid hydrolases
Examples: LDL particle proteins and lipids hydrolyzed by lysosomal proteases and esterases, respectively + Free amino acids for protein synthesis and cholesterol used for membrane synthesis
What are the steps for the delivery of membrane proteins to the lysosome interior for degradation?
During receptor-mediated endocytosis, ‘free’ receptors recycled back to cell surface OR delivered to lysosome interior for degradation
Cell-surface receptors destined for degradation/turnover due to damage or control of cellular signaling pathway (receptors for insulin (hormones) and epidermal growth factors (EGFs))
Degradation of endocytosed membrane proteins (e.g. cell surface receptors) involves inward budding of vesicles into late endosome interior forming multivesicular endosome or multivesicular body (MVB)
2) Involves sorting at MVB of nascent lysosomal membrane proteins from TGN (via the biosynthetic pathway of step 1. From membrane proteins destined (via the endocytic pathway (step 1) for degradation in lysosomes
MVB has unique morphology – contains numerous intraluminal vesicles
Similar in size to transport vesicles, but opposite topology: MVB vesicles bud away from the cytoplasm (unlike COPI/COPII and clathrin vesicles – bud toward cytoplasm)
3) MVB vesicles selectively contain membrane proteins destined for degradation in the lysosome interior
Membrane ‘cargo’ protein selection and inward (vesicle) budding mediated by ESCRT machinery
What is ESCRT?
Multi protein complex – soluble protein constitutions recruited to MVB surface to mediate membrane ‘cargo’ protein selection and inward vesicle budding
Endocytosed membrane proteins destined for degradation linked to mono-ubiquitin
Serves as a signal for recognition by ESCRT proteins Hrs and subsequent entry (packaging) into newly- forming MVB vesicle
(step 1): Hrs also mono-ubiquitinated
Poly-ubiquitination = signal for proteasomal degradation (ERAD pathway)
Hrs recruits additional ESCRT proteins
Assembly to mediate inward budding and scission of nascent vesicle into MVB lumen (step 2) = COPI/II and clathrin coat vesicle assembly, but in the opposite direction
Disassembly of ESCRT complex by ATPase Vps4
ESCRT components released into the cytoplasm and ‘recycled’ for additional rounds of MVB formation (step 3)
4) MVB fuses with a lysosome
MVB trafficking/docking/fusion with lysosome mediated by organelle-specific Rabs/Rab effectors & SNAREs
Intraluminal vesicles with endocytosed membrane proteins (i.e., cell-surface receptors destined for turnover) released into the lysosome interior and degraded by acid hydrolases
Lysosomal membranes in MBV (boundary) membrane (trafficked from TGN via biosynthetic pathway) move laterally into the lysosomal membrane
Carry out function(s) and protected from degradation by their attached carbohydrates (glycosylation)
Describe HIV and ESCRT
ESCRT is hijacked by retroviruses for virus budding from the plasma membrane
Topology of vesicle budding at MVB similar to enveloped retrovirus (e.g., HIV) particle budding at the plasma membrane in virus-infected cells (i.e., budding away from cytosol –> away from MVB)
HIV Gag protein functions similar to ESCRT Hrs
Major structural viral particle protein at pm is mon-ubiquitinated and, like Hrs at MVBs, subsequently binds to and recruits (hijacks) ESCRT machinery to the plasma membrane
ESCRT mediates budding and scission of nascent (pm enveloped) virus particle into extracellular space for subsequent infection of other cells
