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32 Cards in this Set
- Front
- Back
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Physiological Functions of Endocytosis
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- bacteria and protein aggregates
- bulk uptake of proteins such as albumin - internalization of different ligands mediated by plasma membrane receptors such as peptide hormones including insulin, prolactin, growth hormone, EGF, etc |
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Types of Endocytosis
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1. Phagocytosis - specialized cells only
2. Macropinocytosis 3. Clathrin mediated endocytosis 4. Pinocytosis 5. Autophagy 6. Caveolae mediated endocytosis, lipid raft endocytosis |
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Adsorptive (receptor-mediated) Endocytosis
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- receptor on cell surface bind ligand
- coated pit formation - endocytosis of receptor ligand complex - uncoating and fusion with Early Endosome - dissociation at low pH - receptor recycled - ligand moves onto late endosome and lysosome - degradation |
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Endocytic Vessicles
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1. Clathrin
2. Non-clathrin: same endosomes as clathrin, cannot concentrate cargo, important for raft domains 3. Phagosomes 4. Caveolae mediated |
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Early Endosome
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- mildly acidic - proton pump - pH 5.5
- recycling - receptor ligand detachment - ligand continues to lysosome - receptor goes back - around 10 minutes - sorted into tubular extensions that detach |
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Multi-vesicular Bodies
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- no more recycling endosomes
- internalized vesicles with proteins for degradation - protein composition different: some degradative enzymes - acidic pH - proteins cycle between MVB and Golgi |
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Lysosome
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- pH 5
- degradative enzymes: saposin, acid phosphates, cathepsin D) - only function at low pH - protected by LAMPs and LIMPs |
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Lysosome Products
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1. nucleases: nucleic acids
2. proteases: specific and non specific 3. glycosidases: glycoproteins, sugar polymers 4. lipases: fatty acids, phospholipids, sphingolipids 5. phosphatases: remove phosphate 6. protective membrane proteins: integral proteins - rest soluble 7. proton pump (also found in early, late endosomes and golgi) 8. membrane hydrolase LAP 9 activator proteins: extracting lipids from membrane - needed for lipase function |
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Prevention of Acidification
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1. Weak bases
- ammonium chloride - chloroquine: enters and grabs protons, can accumulate, can result in endosomal suffocation in neurons 2. Lonophores: (monensin) makes pores - protons leak back 3. Bafilomycin (proton pump inhibitor) best choice, no effect on mitochondrial pump |
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Study Endocytosis
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1. Electron microscopy - electron dense tracers
2. Light microscopy 3. Compartmental markers - immunogold/fluorescence 4. Membrane fractionation: endosomes purified |
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Low Density Lipoproteins
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- lipid/cholesterol ether droplets, surrounded by phospholipid monolayer, interal hydrophobic protein
- protein will bind to one LDL receptor - clathrin coated pit with AP2 adaptor - no ARF - AP2 also adaptor for transferrin receptor and others - statins used to reduce cholesterol - LDL doesn't bind AP2 - instead ARH alternate adaptor - capable of interacting with AP1 |
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Adaptor Proteins
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binds cargo and clathrin
ear and hinge: clathrin mu: cargo (includes many receptors via dileucine or tyrosine motifs) |
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Pathway 1
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- LDL, insulin, prolactin
- receptor recycled, ligand degraded - cholesterol for nutritional purposes - insulin for signalling purposes |
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Pathway 2
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- Epidermal Growth Factor (EGF) receptor
- goes into mitosis, divides and differentiates - harmful with a strong signal - receptors dimerize, become ubiquitinated, budding into vesicles - join the MVB - pH - no effect and remain bound to ligand - both are degraded - down regulation |
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Pathway 3
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- diferric transferrin: both receptor and ligand recycled back
- iron, transferrin, receptor bind at neutral pH and are taken to endosomes AP2 adaptor cells - iron comes off at acidic, transferrin and receptor remain bound - iron transported out - transferrin and receptor go back to cell surface - dissociate under neutral pH |
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Pathway 4
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- IgA and IgG - transcytosis (trans epithelial vesicular transport)
- IgA needs to be transported to mucous membrane from blood stream where it is synthesized - Fc receptors that bind to antibody chains - endocytose partially across the epithelia - switches to different receptors and exocytosed halfway into the lumen |
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Macropinocytosis
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- formation of large vacuoles - bending of lamellipodia
- non specific - smaller than phagosome - requires actin - will eventually merge |
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Lipid Rafts
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- more saturated, longer carbon chains, cholesterol - thicker, more rigid membrane - longer TM domains
- GPI proteins (luminal side) sphingolipid anchor - concentrate certain proteins - from trans golgi to apical domain in tubular transport - associate with caveolae |
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GPI
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- clathrin independent endocytosis
- tubular structures internalized, dependent on actin and cdc42 - referred to as GEECs - acidic and fuse with early endosomes (eg: folic receptor) |
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Caveolae
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- coated with caveolin (integral membrane protein)
- cholesterol rich membrane concentrated in caveolae - neutral pH compartments called caveosomes - transcytosis of albumin across endothelium - very rapid process - remain at the cell surface - signalling platform - not essential but efficient |
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Caveolae Mediated Transcytosis
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- endothelial cells of capillaries - thin forming tight seal
- caveolae bud off at random - transport serum proteins - no strong concentration of cargo - 3 types: 1 and 2 everywhere, 3 mainly in muscles - hairpin loops in and out - no uncoating/budding |
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Targeting to Lysosome - Method 1 - Mannose 6 Phosphate Receptor
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- hydrolase has N linked oligosaccharide - sit in GlcNAc recognition site
- GlcNAc removes N-acetylglucosamine - exposes M6P signal (cis compartment) - signal stays till trans golgi where it binds to M6P receptor - recognized by clathrin coat - vesicle - endosome: cargo goes to lysosome, receptor recycled but NOT through clathrin - GGA adaptor proteins used |
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Targeting to Lysosome - Method 2 - Sortilin
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- receptor that uses GGA adaptor proteins
- transport of activator protein, prosaposin - binds cargo and outside binds clathrin |
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Prosaposin
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- activator protein transported by sortilin
- binds to sortilin and GGA - chopped up into 4 saposins - activators - degrade lipids - important in degradation of sugars to Ceramide - diseases result in glycolipid build up: a. Gauchers: mental retardation and death in young age. lysosomal storage disease b. Tay-Sachs disease: |
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GGA
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- adaptor protein - binds on trans Golgi
- bind to receptor tail motif via VHS domain (M6P and sortilin) - receptor tail targeted by acidic dileucine cluster - binds ARF1 via GAT domain - binds clathrin via hinge and terminal ear domain - truncated: doesn't bind clathrin, dominant negative since can interact with ARF1 and stop process |
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Transport of Lysosomal Membrane Proteins
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- LEP100, LIMP1-4, LAMPS are membrane glycoproteins
- golgi to the lysosome using adaptor proteins - LAMP by AP3 - mu to cargo, ear/hinge to clathrin - bind to receptor tails via tyrosine or dileucine - short cytoplasmic domains unlike trasferrin - AP3 binds to tyrosines close to the membrane - without mu3 in AP3 - process slower as it goes to the cell membrane and then back through AP2 |
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Transport of Cytoplasmic Proteins
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- lysosome from cytoplasm
- ribonuclease A has a KFERQ motif - binds to hsp prior to lysosome delivery |
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Retromer
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- transport of M6P and sortilin receptors from late endosome to the golgi
- tubular buds |
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Reasons for Autophagy
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1. removal of protein aggregates
2. cell starvation 3. bacterial or infectious agent 4. defective organelles 5. specialized purposes (cholesterol from lipid droplets) |
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Autophagy
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- not endocytic process
- ubiquitination of substances too large for chaperonal degradation |
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Kinds of Autophagy
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1. Chaperone mediated: KFERQ tagged proteins - bind to hsp70 - translocated to lysosomes via LAMP2A
2. microautophagy: proteins/hsp70 adhering to phosphatidylserine on surface of late endosomes budded into internal vesicles |
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Macroautophagy
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- formation of phagophore membrane
- cleavage of LC3 and conjugation with PE and LC3 and insertion into phagophore membrane to form LC3II (irreversible - degraded eventually) - LC3 on membrane: will fuse with phagosome - phagophore membrane extends around organelle - produce early autophagosome - target ubiquitinated - P62 adaptor binds ubiquitin and LC3 forming a bridge between phagophore membrane and target - autophagosome exchanges membrane with endosomes - fuses with lysosome - contents degraded - internal membrane degraded with lysosome fuses |
