05_Protein_Trafficking_2024.07.26_Student.pptx

Protein Trafficking Steven J. Ontiveros, MBA, PhD  Objectives Describe the composition of the nuclear envelope, explain the mechanism of nuclear import by nuclear pore complexes with the aid of GTPases, and nuclear localization signals responsible for targeting. D...

Protein Trafficking Steven J. Ontiveros, MBA, PhD  Objectives Describe the composition of the nuclear envelope, explain the mechanism of nuclear import by nuclear pore complexes with the aid of GTPases, and nuclear localization signals responsible for targeting. Describe the mechanism of vesicle budding and vesicular transport within the secretory and endocytic pathways. Explain the major transport mechanisms, the roles signal sequences play in organelle targeting. Explain the mechanism of receptor mediated endocytosis during the uptake of selective molecules. Explain the mechanisms that target proteins to the endoplasmic reticulum and how proteins are inserted into the membrane or lumen after protein translation. Explain the types of protein modifications that occur in the Golgi complex and roles it plays in the secretory pathway.  Protein Sorting and Transport Organelles Membrane-enclosed Unique compartments with unique functions Specialized spaces within the cell Cells must: Synthesize unique proteins Transport proteins to the appropriate place Cell Biology 3rd Edition, 2017 3  Protein Sorting and Transport Pathways 3 Major types of pathways: A. Endocytic pathway Inward movement of cargo (endocytosis) Ingestion of extracellular molecules B. Secretory/Exocytic pathway C. Intracellular Outward movement of cargo (exocytosis) path Sends molecules out of the cell C. Intracellular transport pathway Movement of cargo within cell and between cellular compartments/organelles Cell Biology 3rd Edition, 2017 4  Ribosomes and Protein Synthesis Protein synthesis Takes place on ribosomes 2 Types of Ribosomes 1. Free ribosomes In the cytosol These are proteins destined for the nucleus, mitochondria, peroxisomes 2. Membrane (ER) bound ribosomes Proteins destined for the ER, Golgi, endosomes, lysosomes, nuclear membrane, plasma membrane, and secretion Junquiera’s Basic Histology 15th Edition, 2018 ER Protein synthesis Most animal cell protein is made in ER 5  Sorting Signals Sorting Signals: Amino acid sequence that directs protein targeting Ex: ER retention sequence Lys-Asp-Glu-Leu (KDEL) Function of Sorting Signals: Direct proteins to their correct destination (molecular address) Sorting Signal sends protein to appropriate organelle/cellular space No signal, protein heads to the cytosol 6 Endoplasmic Reticulum Synthesis and Targeting 7  ER Protein Synthesis Histology and Cell Biology 5th Edition, 2020 Two major components for ER synthesis Signal recognition particle (SRP) Binds ER signal sequence SRP Receptor (bound to ER membrane) Binds SRP Mechanism: SRP recognizes the ER signal sequence SRP facilitates binding of ribosome complex to SRP receptor (SRPr) Polypeptide is inserted into membrane channel Translocation of polypeptide is driven protein synthesis Polypeptide is released into ER lumen 8  ER Export and ERGIC ER Exit Site (ERES) Specialized region for vesicle budding Proteins that contain ER export signals leave the ER Bidirectional vesicular transport ER and the Golgi complex Anterogra Transport types de Retrograde Anterograde – ER  Golgi (COPII coats) Retrograde – Golgi  ER (COPI coats) Endocytic/Exocytic - Use clathrin coats ER Cell Biology 3rd Edition, 2017 9  Vesicles for Protein Transport Transport of proteins uses vesicles Vesicle budding Budding from source (sending) organelle Vesicle fusion Vesicles containing protein fuse with target organelle Vesicles use cytoskeletal elements for transport 10  Clathrin Coats and Vesicle Budding Vesicle Protein coat Budding membranes (ER, Golgi, Plasma membrane) have protein coat Clathrin coated vesicle budding Golgi budding (outward) Plasma membrane budding (inward) Vesicle formation 1. Binding: Adaptin binds cargo receptor 2. Recruitment: Clathrin is recruited to form coat 3. Maturation: Clathrin drives budding 4. Scission: Clathrin coat is pinched off by dynamin ring (GTP-binding protein) Clathrin eventually uncoats allowing vesicle to fuse with target 11  Vesicle Targeting & Fusion Rab Protein Rab1 Transport Step ER to Golgi Rab3 Exocytosis Budding (sending) and Fusion Rab4 Endosome to PM (delivery) Rab7 Endosome to lysosome Highly regulated system Ensures delivery of proper cargo to correct destination Rab proteins – mark transport vesicles (molecular address) Targeting and docking Vesicle is transported by motor proteins Targeting is established by Rab proteins Address is recognized by tethering proteins SNAREs provide additional specificity (v- and t-SNARES) 12 Transport to Golgi Complex 13  Protein Sorting: The Golgi Apparatus Golgi: Modifies proteins Sorts/organizes proteins Packages proteins into vesicles 3 Major compartments Cis (cGolgi) Medial (mGolgi) Trans (tGolgi) 14 Transport Pathways from Golgi 3 major pathways from the Golgi 1. Golgi to other organelles (ER/endosomes/lysosomes) 2. Golgi to cell membrane Direct transport Continuous secretion of protein 1 3. Regulated secretion Hormones held in immature secretory 3 2 granules Environmental signal causes release 15 ER Synthesized Protein Lysosome Targeting 16  Lysosome Targeting ER Protein Synthe sis Medical Physiology 3rd Edition, 2017 17  Lysosome Targeting ER to Golgi to Endosomes to Lysosomes ER  Golgi  Lysosomes Lysosomal enzymes Mannose-6 is phosphorylated (M6P) in cGolgi M6P receptor (tGolgi) binds M6P residues tGolgi sends proteins to endosomes Medical Physiology 3rd Edition, 2017 18  Lysosome Formation Lysosomal acid hydrolases Proteins (hydrolases) are transported to endosomes from tGolgi Clathrin-coated vesicles fuse with late endosomes Accumulation of hydrolases Acidic environment (pH ~ 5.5) causes proteins to dissociate Medical Physiology 3rd Edition, 2017 from M6P receptor Endosomes mature into lysosome 19 Cytosolic Protein Synthesis Nuclear Import 20  Nuclear Import Cytosolic (free) ribosomes Synthesize nuclear proteins Nuclear pore Gate – mediates selective nuclear import/export Nuclear localization signal (NLS) Facilitates nuclear import Proteins with NLS are imported by nuclear receptors 21  Mechanism of Nuclear Import Importins: Recognize nuclear localization signal and carry proteins into nucleus Work in conjunction with GTP-binding protein Ran Mechanism: Import Importin recognizes NLS on cargo in protein Importin interacts with pore complex and imports protein into nucleus Ran-GTP bind importin  displaces protein Cargo protein is released into nucleus Ran-GTP and Importin complex exit nucleus Hydrolysis of GTP  GDP on Ran Ran-GDP releases importin Importin binds a new cargo protein with NLS 22 Histology and Cell Biology 5th Edition 2020 Endocytic Pathway 23  Endocytic Pathway Phagocytosis (eating) Ingestion of large particles, microorganisms, or other cells Macrophages Pinocytosis (drinking) Ingestion of fluid and small molecules 24  Receptor Mediated Endocytosis Selective uptake: Metabolites, hormones, proteins, and viruses Molecule binds receptor Ex: Cholesterol Hydrophobic Transported as an LDL Mechanism: Histology and Cell Biology 5th Edition Ligand binds receptor Ex: LDL binds LDL receptor Internalized via clathrin coated mechanism Material is degraded via 2020 lysosomes and recycled 25 26