Endocytosis and Lysosome Formation The Endolytic Pathway.

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– 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• AP1, 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

• AP2-receptor interaction

• Receptor clustering• Clathrin recruitment• Dynamin collar

assembly• Budding• Uncoating & recycling of components

Coated Pit Targeting Sequences

• FxNPxY

• Yxx• Tyrosine residue in these contexts is

necessary & sufficient to mediate AP2 interactions and subsequent clathrin assembly

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

CalveolaeOr 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

• pH 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