Vesicular Traffic, Secretion, and Endocytosis PDF

1 VESICULAR TRAFFIC, SECRETION, AND ENDOCYTOSIS 10/20/2024 Tobias Weinrich, PhD School of Integrative Biological and Chemical Sciences  2 Student Learning Outcomes ▪ Reading guide learning objectives ▪...

1 VESICULAR TRAFFIC, SECRETION, AND ENDOCYTOSIS 10/20/2024 Tobias Weinrich, PhD School of Integrative Biological and Chemical Sciences  2 Student Learning Outcomes ▪ Reading guide learning objectives ▪ Relate the structure of the Golgi apparatus to its function. ▪ Describe the types of protein glycosylation that take place in the Golgi. ▪ Diagram the routes of protein export from the Golgi. ▪ Summarize the process of vesicle budding and cargo selection. ▪ Explain the role of coat proteins. ▪ Describe the mechanism by which vesicles fuse with the correct target membranes. ▪ Describe the function of lysosomes. ▪ Explain how lysosomes are formed. ▪ Describe the various forms of endocytosis (e.g., phagocytosis, pinocytosis, receptor- mediated endocytosis) and their roles in cellular uptake. ▪ Summarize the pathway of clathrin-mediated endocytosis. ▪ Explain recycling of cell surface receptors. ▪ Differentiate between constitutive and regulated secretion pathways  3 Lecture Structure 1. Golgi Apparatus 1.1. Function 1.2. Structure and Composition 1.3. Glycosylation 1.4. Sorting 2. Principles of Vesicular Transport 2.1. Coat Proteins 2.2. Mechanisms of Vesicle Budding 2.3. Mechanisms of Vesicle Docking and Fusion 3. Early stages of Vesicular Transport 4. Late stages of Vesicular Transport A. Lysosomes B. Endocytic Pathway C. Secretory Pathway  4 1.1. Golgi Apparatus - Function FUNCTION a) Terminal Protein glycosylation: ▪ Modification of N-linked oligosaccharides ▪ Complex polysaccharides ▪ High-mannose oligosaccharides ▪ Addition of O-linked oligosaccharides ▪ Proteoglycans b) Glycolipids and sphingomyelin synthesis ▪ Ceramide precursor c) Sorting of lipids, proteins, and polysaccharides: ▪ Endosomes & Lysosomes ▪ Plasma membrane ▪ Secretion  5 1.2. Golgi Apparatus – Structure and Composition ▪ Flattened membrane-enclosed sacs(cisternae) and associated vesicles ▪ Polarity: ▪ cis (entry) – oriented towards the ER/nucleus ▪ medial ▪ trans (exit) – oriented towards the cell membrane ▪ Transport vesicles ER ▪ Proteins are modified and sorted as they pass through the different Golgi compartments Plasma membrane  6 1.2 Golgi Apparatus – Structure and Composition Movement of lipids and proteins from the CGN to the TGN: A. Cisternal Maturation Model – cisternae gradually change in composition as they themselves move forward B. Vesicle Transport Model – shuttle vesicles carry material forward from the ER to successive Golgi compartments  7 1.3 Golgi Apparatus – Glycosylation A. Modification of N-linked oligosaccharides ▪ Trimming of mannose (Man) ▪ Except in high-mannose oligosaccharides ▪ Addition of complex oligosaccharides: ▪ N-acetylglucosamine (GlcNAc) ▪ Sialic Acid (NANA) ▪ Galactose B. Addition of O-linked oligosaccharides ▪ Serine and threonine (O-atom) ▪ Sequential addition of single sugar residues ▪ Short ▪ Long (>200 residues) = proteoglycans GlcNAc // Galactose // NANA  8 1.3 Golgi Apparatus – Glycosylation EXAMPLE: Lysosomal proteins modification ▪ Sorting to lysosomes – Mannose-6-P (M6P) 1. GlcNAc phosphotransferase transfers phosphate group 1 2 2. GlcNAc glycosaminidase removes terminal GlcNAc ▪ M6P receptor ▪ Example: acid hydrolases ▪ I-cell disease – absent GlcNAc phosphotransferase  9 1.4 Golgi Apparatus – Sorting Sorting of proteins (soluble and membrane proteins), lipids, and carbohydrates. A. Lysosomes B. Endocytic – endocytic vesicles ▪ Endocytosis – uptake of materials from the exterior of the cell ▪ Receptor-mediated endocytosis ▪ Pinocytosis Receptor sorting in endosome: lysosomes, recycling, transcytosis ▪ Phagocytosis C. Secretory Pathways – secretory vesicles Protein sorting in TGN: ▪ Regulated secretion – controlled, rapid releases that happen in response to a signal ▪ Constitutive secretion – continuous discharge of vesicles at the plasma membrane  10 2. Principles of Vesicular Transport Vesicular transport – transport vesicles bud off from one compartment and fuse with another ▪ Membrane ▪ Lumen ▪ Cargo 1. Secretory Pathway ▪ ER → Golgi → Plasma membrane (exocytosis) ▪ ER → Golgi → Endosomes → Lysosomes 2. Endocytic Pathway ▪ Plasma Membrane (endocytosis) → Endosomes → Lysosomes 3. Retrieval/recycling ▪ Recycling of membranes and receptors  11 2.1. Coat Proteins ▪ Coat proteins – facilitate: ▪ Cargo selection ▪ Budding of vesicles ▪ Coat proteins disassemble prior to fusion with target membrane TYPES: A. Clathrin ▪ AP1: Golgi → Lysosomes ▪ AP2: Plasma membrane → Endosomes B. COPI ▪ Golgi → ER C. COPII ▪ ER → Golgi Unknown coat protein for secretory pathways  12 2.2. Mechanisms of vesicle budding Monomeric G protein 1. Recruitment – adaptor proteins, receptors, v-SNARE, cargo protein (sorting signal), Rab (monomeric G protein) Coat-recruitment GTPases: ▪ Clathrin and COPI coated vesicles – ARF monomeric G protein ▪ COPII coated vesicles – Sar1 monomeric G protein 2. Polymerization of coat proteins + membrane invagination/curvature 3. Vesicle Budding ▪ Dynamin – pinches off vesicle bud in clathrin coated proteins 4. ARF/Sar1 GTP hydrolysis → coat disassembly  13 2.2. Mechanisms of vesicle budding EXAMPLE: Clathrin-coated vesicles ▪ Cargo receptor – recognize cargo molecules ▪ Adaptor protein (AP1/2) – coat protein that binds to cargo receptor ▪ Clathrin triskelion polymerization – clathrin coat ▪ ARF small G-protein ▪ Vesicle formation (budding) – dynamin ▪ Coat shedding – ARF GTP hydrolysis  14 2.3. Mechanisms of vesicle docking and fusion 1. Docking/tethering – Rab-GTP binding to effector protein on target membrane 2. Fusion – v-SARE (vesicle membrane) binding to t-SNARE (target membrane) ▪ Facilitated by Rab-GTP hydrolysis ▪ Dissociation of SNARE complex by NSF + ATP-hydrolysis  15 2.4. Specificity of vesicle transport ▪ Phosphoinositide – membrane identity ▪ Different phosphoinositides (like PI(4)P, PI(3)P,…) are found in distinct membranes ▪ Recruitment of specific proteins required for vesicle formation ▪ Recruitment of specific Rab-effector proteins required for vesicle docking ▪ Rab small-G-proteins – guide transport vesicles to their target Membrane  16 3. Early Stages of Vesicular Transport ▪ COPII: ER → Golgi (anterograde transport) ▪ COP I: Golgi → ER (retrograde/retrieval transport) ▪ Missorted ER resident proteins – KDEL sequence  17 4. Late Stages of Vesicular Transport Sorting of proteins (soluble and membrane proteins), lipids, and carbohydrates. A. Lysosomes B. Endocytic – endocytic vesicles ▪ Endocytosis – uptake of materials from the exterior of the cell ▪ Receptor-mediated endocytosis ▪ Pinocytosis Receptor sorting in endosome: lysosomes, recycling, transcytosis ▪ Phagocytosis C. Secretory Pathways – secretory vesicles Protein sorting in TGN: ▪ Regulated secretion – controlled, rapid releases that happen in response to a signal ▪ Constitutive secretion – continuous discharge of vesicles at the plasma membrane  18 4. Late Stages of Vesicular Transport A. LYSOSOMES ▪ Single membrane bound organelles - 0.5 μm in diameter ▪ FUNCTION – intracellular digestion ▪ Acid hydrolases (hydrolytic enzymes) – active at pH~5 ▪ Avoid uncontrolled digestion in other compartments ▪ Sorting signal: Mannose-6-Phosphate ▪ Coat: clathrin ▪ Retrieval pathway: recycling of M6P receptor  19 4. Late Stages of Vesicular Transport A. LYSOSOMES ▪ FORMATION – multiple pathways deliver material to lysosomes ▪ Endocytosis → early endosomes → late endosomes ▪ Macropinocytosis ↓pH (V-H+pump) → Lysosomes ▪ Phagocytosis → phagosome Acid Hydrolases ▪ Autophagy → autophagosome  20 4. Late Stages of Vesicular Transport B. ENDOCYTOSIS ▪ Endocytic vesicles ▪ Processes: ▪ Receptor mediated endocytosis (selective) Clathrin dependent ▪ Pinocytosis (clathrin-coated pits) ▪ Caveolae ▪ Macropinocytosis Clathrin independent ▪ Phagocytosis → phagosome ▪ ENDOSOME MATURATION – early endosomes (beneath plasma membrane) – mature into late endosomes (closer to Golgi) ▪ Fuse with lysosomal vesicles from Golgi → lysosomes ▪ Recycling endosome ▪ Redistribution to other membranes ▪ Recycling of plasma membrane components  21 4. Late Stages of Vesicular Transport B. ENDOCYTOSIS – Receptor mediated endocytosis EXAMPLE: cholesterol and LDL receptor mediated endocytosis ▪ LDL Receptor – bind ligand ▪ Clathrin coated pits ▪ Clathrin ▪ Adaptor Protein (AP2) ▪ What is degraded? ▪ What is recycled?  22 4. Late Stages of Vesicular Transport B. ENDOCYTOSIS – Receptor mediated endocytosis ▪ Fate of receptors: ▪ Degraded in lysosome ▪ Recycling endosomes: ▪ Recycling to original membrane (e.g. LDL-receptor) ▪ Transcytosis – redistribution to other membranes (e.g. Fc receptors)  23 4. Late Stages of Vesicular Transport C. SECRETORY PATHWASY ▪ Default pathway ▪ Secretory vesicles Pathways: 1. Constitutive secretion (all cells) – continuous discharge of vesicles at the plasma membrane 2. Regulated secretion  24 4. Late Stages of Vesicular Transport C. SECRETORY PATHWASY 2. Regulated secretion (specialized secretory cells) – controlled, rapid releases that happen in response to a signal – released in response to hormone/neutrotransmitter, Ca2+ a) Secretory vesicles – formed in TGN ▪ Cargo concentration, storing, and processing (e.g. proteolytic cleavage) of secretory proteins PM PM Golgi Golgi Proinsulin → protein cleavage → insulin b) Synaptic vesicles (nerve cells) – formed from recycling endosomes ▪ Concentration of neurotransmitters  25 4. Late Stages of Vesicular Transport C. SECRETORY PATHWASY 2. Regulated secretion (specialized secretory cells) – controlled, rapid releases that happen in response to a signal – released in response to hormone/neutrotransmitter, Ca2+ a) Secretory vesicles b) Synaptic vesicles (nerve cells) – formed from endosomes ▪ Neurotransmitter is loaded in synaptic terminal ▪ Concentration of neurotransmitters  26 4. Late Stages of Vesicular Transport C. SECRETORY PATHWASY Selective transport from TGN in polarized cells (e.g. epithelial cells) ▪ Sorting of membrane proteins and lipids occurs in the TGN ▪ Apical membrane ▪ Basolateral membrane ▪ Direct sorting (A) ▪ Indirect sorting via endosomes (transcytosis) (B)