Exocytosis, Endocytosis, Endosomes and Lysosome Formation The Endolytic Pathway

Cargo Sorting by the Trans-Golgi Network (TGN) Specific coat proteins direct TGN vesicles to targets

Proteolytic Processing of Pro-proteins in Secretory Vesicles Cleavage of proinsulin in secretory vesicles of pancreas cells

Secretory Vesicles/Granules Material in secretory vesicle aggregates due to decreased pH & divalent cations Membrane recycling to TGN directly or via endosome reduces vesicle size

Regulated Secretion Fusion of the vesicle and plasma membrane is regulated by various means receptor-ligand interaction and second messenger signaling depolarization of membrane etc…

Lysosomal Targeting of Prohydrolase by M6P and MPRs TGN lumen Mannose-6-Phosphate Receptors Cation-independent (CI) Cation dependent (CD) cytosol

MPR Cytoplasmic Tail Routing Signals

Assembly of Clathrin on Lysosomal/Endosomal Destined TGN Vesicles

Lysosomal/Endosomal Targeting MPR targeting to lysosome/endosome bound vesicles Vesicle Coats AP1 Adaptor proteins -  &  adaptins GGA Golgi-localising, gamma-adaptin ear domain homology, ARF-binding proteins AP3  &  adaptins AP2 exclusive to plasma membrane  &  adaptins

Adaptor Protein Components

Directed Translocation of Secretory Vesicles apical targeting in caveolin coated, lipid-raft vesicles basolateral targeting signals direct protein to this membrane constitutive basolateral targeting subsequent sorting of apical proteins untargeted vesicles sorting to redistribute surface proteins tight junctions E-cadherin tight junctions syntaxin-3

Endocytic Mechanisms

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

Lysosome Structure MVBs 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

Multivesicular Bodies Vps – vacuolar protein sorting (yeast genes) ESCRT – endosomal sorting complexes required for transport

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

Rab GTPases Regulate Endocytic Membrane Interactions Ras related GTPase from rat brain

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

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

Ok, It's In the Cell. Now What pH dependent interactions affects protein-protein interactions in endolytic compartments Acidification mediated by V1V0 H+ ATPase

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

Clathrin

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

Coated Pit Targeting Sequences FxNPxY LDL receptor YTRF Transferrin receptor Yxx minimum consensus Tyrosine residue in these contexts is necessary & sufficient to mediate AP2 interactions and subsequent clathrin assembly

Targeting Sequences

Endocytosis Kinetics Bulk rate Binding-studies & Turnover LDLR EGFR CD4 channels Endocytosis Kinetics constitutive 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 Ligand induced Ligand dependent anchor release resident

Calveolae Plasmalemmal vesicles Associated with lipid rafts 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

Phagocytosis

Macropinocytosis in Action

Invasive Particles Evade Degradation