1. Endocytosis and Lysosome Formation The Endolytic Pathway

2. Lysosomes and Endosomes Early endosome –pH 6.5-6 –sorting endosome Late endosome –pH 5.5-5 Lysosome –pH 5-4.5

3. 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

4. Phagocytosis

5. Endocytic Mechanisms cytockeletal element involvement –macropinocytosis –phagocytosis clathrin coated pits calveolin coated calveolae non-coated pit pinocytosis

6. Receptor-Mediated Endocytosis Clathrin-coated pits AP1, 2, 3, 4 –heterotetramer –  & γ subunits target –  subunits bind clathrin –  binds receptor signal sequence clathrin triskelion –hexamer of 3 heavy & 3 light chains

7. Formation of Coated Pit AP2-receptor interaction Receptor clustering Clathrin recruitment Dynamin collar assembly Budding Uncoating & recycling of components

8. Coated Pit Targeting Sequences FxNPxY Yxx  Tyrosine residue in these contexts is necessary & sufficient to mediate AP2 interactions and subsequent clathrin assembly

9. 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 LDL EGFR CD4 Pumps

10. 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

11. Calveolae Calveolin –high-affinity cholesterol binding protein

12. Macropinocytosis in Action

13. Pretty Colored Vesicles Gold dust through the endolytic pathway –coated pits (CP) –early endosome (EE) –late endosome (LE) –lysosome (LY)

14. Recycling Membrane Components from Endosomes Tubulovesicular morphology –membrane concentrated tubules –bud & recycled directly & indirectly

15. 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

16. Some Examples of Receptor Mediated Endocytosis Receptor recycling –LDL-LDLR –transferrin-transferrin receptor Receptor destruction –EGF-EGFR