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Endocytosis and Lysosome Formation. The Endolytic Pathway. Lysosomes and Endosomes. Early endosome pH 6.5-6 sorting endosome Late endosome pH 5.5-5 Lysosome pH 5-4.5. Structure of Lysosomes. Protein rich membranes Lamp/Limp family of glycoproteins Unusual lipids

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lysosomes and endosomes
Lysosomes and Endosomes
  • Early endosome
    • pH 6.5-6
    • sorting endosome
  • Late endosome
    • pH 5.5-5
  • Lysosome
    • pH 5-4.5
structure of lysosomes
Structure of Lysosomes
  • Protein rich membranes
    • Lamp/Limp family of glycoproteins
  • Unusual lipids
    • lyso-bisphosphatidic acid
    • thought to protect lysosomal membrane lipids from action of lumenal lipases
  • Multivesicular bodies (late endosomes/lysosomes)
    • invagination of the limiting membrane
    • forms internal membranes
      • TM proteins can segregate into limiting or internal membranes
      • limiting membrane can be recycled, internal is not
endocytic mechanisms
Endocytic Mechanisms
  • cytockeletal element involvement
    • macropinocytosis
    • phagocytosis
  • clathrin coated pits
  • calveolin coated calveolae
  • non-coated pit pinocytosis
receptor mediated endocytosis
Receptor-Mediated Endocytosis
  • Clathrin-coated pits
  • AP1, 2, 3, 4
    • heterotetramer
    •  & γsubunits target
    •  subunits bind clathrin
    •  binds receptor signal sequence
  • clathrin triskelion
    • hexamer of 3 heavy & 3 light chains
formation of coated pit
Formation of Coated Pit
  • AP2-receptor interaction
  • Receptor clustering
  • Clathrin recruitment
  • Dynamin collar assembly
  • Budding
  • Uncoating & recycling of components
coated pit targeting sequences
Coated Pit Targeting Sequences
  • FxNPxY
  • Yxx
  • Tyrosine residue in these contexts is necessary & sufficient to mediate AP2 interactions and subsequent clathrin assembly
endocytosis kinetics

LDL

EGFR

CD4

Pumps

Endocytosis Kinetics
  • Bulk rate
    • measurement of lipid movement
  • Binding-studies & Turnover
    • experiments designed to measure ligand-receptor interaction strength and rate of receptor/ligand destruction, synthesis and replacement
calveolae
Calveolae

Or as they're better known…

  • Plasmalemmal vesicles
  • Associated with lipid rafts
  • Membrane lipid cycling
  • Raft-associated protein endocytosis/exocytosis
    • Inner leaflet proteins
      • acylated proteins
      • prenylated proteins
    • Outer leaflet proteins
      • GPI-linked
    • Some TM proteins that interact specifically with glycosphingolipids/cholesterol
calveolae11
Calveolae
  • Calveolin
    • high-affinity cholesterol binding protein
pretty colored vesicles
Pretty Colored Vesicles
  • Gold dust through the endolytic pathway
    • coated pits (CP)
    • early endosome (EE)
    • late endosome (LE)
    • lysosome (LY)
recycling membrane components from endosomes
Recycling Membrane Components from Endosomes
  • Tubulovesicular morphology
    • membrane concentrated tubules
    • bud & recycled directly & indirectly
ok it s in the cell now what
Ok, It's In the Cell. Now What
  • pH dependent interactions
    • affects protein-protein interactions in endolytic compartments
  • Acidification mediated by H+ ATPase
    • vacuolar ATPase
some examples of receptor mediated endocytosis
Some Examples of Receptor Mediated Endocytosis
  • Receptor recycling
    • LDL-LDLR
    • transferrin-transferrin receptor
  • Receptor destruction
    • EGF-EGFR