Lecture 11 Vesicular Trafficking: Lysosomes; endocytosis and exocytosis From cell exterior to lysosome What’s taken in? Macromolecules, particulate substances, other cells! Process: a small portion of the plasma membrane, invaginates,pinch off and form “endocytic vescile Tow typpes based on size: phagocytosis (phagosome)--cellular eating! >250 nmAnd pinocytosis (pinocytic vescicles)--cellular drinking! 100 nm

macromolecules are degraded in lysosomes pH is one of the mechanisms for protecting contents in the cytosol All major classes of macromolecules are degraded in lysosomes 40 hydrolytic enzymes Lysosomal glycoproteins are unusually highly glycosylated Vacuolar H+ ATPase: Lysosomes Endosomes Selected compartments of Golgi Apparatus Transport vesicles Secreted vesicles

Lysosomes are highly heterogeneous Shape and size But all have acid hydrolases The “stomach” of a cell Stained for acid phosphatase: Phosphatase substrate

Huge vacuoles of plant cells Related to lysosomes Storage organelle Degradative compartments Turgor pressure Homeostatic device Rubber Opium Garlic Proteins Pigments Noxious molecules

Three pathways to degradation in lysosomes pH 6.0 pH 5.0

The structure of mannose 6-phosphate on a lysosomal enzyme A unique marker for lysosomal proteins

The transport of newly synthesized lysosomal hydrolases to lysosomes M6P receptor pH6.0 pH6.5-6.7 Recycling of M6P

A signal patch in the hydrolase polypeptide chain provides the cue for M6P addition Defects in the GlcNac phosphotransferase cause a lysosomal storage disease in humans, inclusion-cell Disease (I-cell disease) N-acetylglucosamine Lysosomal storage diseases Some lysosomes may undergo exocytosis

Phagocytosis by a macrophate Phagocytosis by a neutrophil pseudopods 5 um cell! Professional phagocytes: macrophages, neutrophils, dendritic cells Phagocytosis is a triggered process, need receptor recognition and signaling; antibodies are good triggers, Fc region, neutriphil and bacteria Pinocytosis is constitutive phosphatidylserine is a trigger for phagocytosis of the dead cell Phagocytosis of inanimated particles: glass, latex beads, asbestos fibers, Living cells:don’t-eat-me signal, inhibitory receptors recrcuit tyrosine phosphatases that antagonize the intracellular signaling events required to initiate phagocytosis Pinocytosis: cell drinking; Phagocytosis: cell eating

clathrin-coated vesicles from the plasma membrane The formation of clathrin-coated vesicles from the plasma membrane 100% plasma membrane turn over in half an hour Endoytic-exocytic cycle Clathrin-coated pits Shed coat and fuse with early endosomes

Caveolae in the plasma membrane of fibroblast Lipid rafts caveolin Non-calthrin coated: caveolae(little cavities) Form from lipid rafts, rich in cholesterol, glycosphingolipids, GPI-anchored membrane proteins Caveolin Caveolae pinch off by virtue of the lipid composition of the membrane Endosome-like compartments or transcytosis Virus entry

A low-density lipoprotein (LDL) particle Receptor-mediated Endocytosis Example: cholesterol uptake 3,000 Kd 1500 cholesterol Molecules 800 phospholipid 500 unesterified cholesterol Atherosclerotic plaques; When uptake is blocked Receptor-mediated endocytosis Cholesterol take up by LDL LDL particle LDL receptor gene mutation--high cholesterol in blood, artherosclerosis Atherosclerosis Cholesterol esters Are hydrolyzed to Free choleterol in lysosomes 500 Kd

Normal and mutant LDL receptor Common endocytic signal: YXXY (binding to adaptin) But LDL receptor: Asn-Pro-Val-Tyr Coronary artery disease

Possible fates for transmembrane receptor proteins Different Rabs associate with early and late endosomes Recycling: LDL receptor

Receptor-mediated endocytosis of LDL

Sorting of transferrin (red) and opioid receptors (green) in the recycling endosomes Transferrin receptor recycles with its ligand Different Rabs Opioid receptors and EGF receptors Are not recycled but degraded in Lysosomes along with ligand: receptor Down-regulations

Some early endosomes Migrate slowly along MT Toward the cell interior And pinch off Vesicles to form MVBs MVBs may fuse with a late endosomal compartment or they fuse with each other to become late endosome

Lysosomes form when vesicles from trans Golgi network fuse with late endosomes

Receptors and their ligands are fully accessible to digestive enzymes in MVBs Ubiquitin tagging facilitate the uptake of receptors into endocytic vesicles and and sorting into the internal membrane vesicles of MVBs

Transcytosis: transferring macromolecules across ephithelial cell sheets A newborn rat obtains antibodies from its mother’s milk Remember transcelluar transport of glucose? Early endosome to recycling endosome Receptors have sorting signals

Exit of membrane proteins from the recycling endosomes can be regulated

Two early endosomal compartments and one common late endosomal compartment in epethilial cells

Exocytosis: fusion of vesicles with the plasma membrane

Two mechanisms of secretion

Three pathways of protein sorting in the trans Golgi network

Secretory proteins form selective aggregates Secretory proteins become highly concentrated: aggregation and membrane retrieval

Proteins are often proteolytically processed during the formation of secretory vesicles A prohormone In different cells Size of the final products Activity

Regulated exocytosis can be a localized response of the plamsa membrane and its underlying cytoplasm Mast cell secreting histamine all over the cell surface when on a solution of soluble stimulant Localized exocytosis when stimulant is presented from a solid bead

Two types of polarized cells Polarized cells direct proteins from the trans Golgi network to the appropriate domain of the plasma membrane

Two ways of sorting plasma membrane proteins Gut epithelial cells Live cells

Lipid rafts in the trans Golgi network Glycosphingolipids and cholesterol form rafts in the lipid bilayer May mediate sorting of glycosphingolipids and GPI-anchored proteins To the apical plasma membrane

The formation of synaptic vesicles 50 nm

Summary Lysosomes are where most of the intracellular degradation occurs. Formation of lysosomes, sorting into lysosomes; Phagocytosis, pinocytosis, receptor-mediated endocytosis, early endosomes, late endosomes, recycling endosomes; Two types of exocytosis, sorting during exocytosis, synaptic vesicles.