Endocytosis and Lysosome Formation The Endolytic Pathway Lysosomes

Endocytosis and Lysosome Formation The Endolytic Pathway

Lysosomes and Endosomes • Early endosome – p. H 6. 5 -6 – sorting endosome • Late endosome – p. H 5. 5 -5 • Lysosome – p. H 5 -4. 5

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

Phagocytosis

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

Receptor-Mediated Endocytosis • Clathrin-coated pits • AP 1, 2, 3, 4 – – heterotetramer & γ subunits target subunits bind clathrin binds receptor signal sequence • clathrin triskelion – hexamer of 3 heavy & 3 light chains

Formation of Coated Pit • AP 2 -receptor interaction • Receptor clustering • Clathrin recruitment • Dynamin collar assembly • Budding • Uncoating & recycling of components

Coated Pit Targeting Sequences • Fx. NPx. Y • Yxx • Tyrosine residue in these contexts is necessary & sufficient to mediate AP 2 interactions and subsequent clathrin assembly

Endocytosis Kinetics LDL • Bulk rate – measurement of lipid movement EGFR CD 4 Pumps • Binding-studies & Turnover – experiments designed to measure ligand-receptor interaction strength and rate of receptor/ligand destruction, synthesis and replacement

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

Calveolae • Calveolin – high-affinity cholesterol binding protein

Macropinocytosis in Action

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 • Tubulovesicular morphology – membrane concentrated tubules – bud & recycled directly & indirectly

Ok, It's In the Cell. Now What • p. H dependent interactions – affects protein-protein interactions in endolytic compartments • Acidification mediated by H+ ATPase – vacuolar ATPase

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