Slide 1Slide 2Slide 3Slide 4Slide 5Slide 6Slide 7Slide 8Slide 9Slide 10Slide 11Slide 12Slide 13Slide 14Slide 15Slide 16Slide 17Slide 18Slide 19Slide 20Slide 21Slide 22Slide 23Slide 24Slide 25Slide 2601.29.10Lecture 7 - Intracellular compartments and transport IISNAREs are proteins that target vesicles to specific compartmentst-SNARES are on target compartmentsv-SNAREs are on vesiclesSNARE proteins are important for membrane fusion•v-SNAREs and t-SNAREs bind tightly•Complexes bring the two membranes together to promote fusionReview of protein translocation1.The secretory pathway: how newly made lipids, proteins, and carbohydrates are delivered to the cell surface via exocytosis2.Endocytic pathways: how cells take up fluids and particles (large and small) from the extracellular environmentExocytosis & endocytosisMost (or all) proteins are covalently modified in the ER1. Formation of disulfide bonds between cysteine residues of the same polypeptide (intra-molecular) or different polypeptides (inter-molecular)2. Addition of short oligosaccharide side chains (glycosylation)Formation of disulfide bonds occurs within the ER•Oxidation of pairs of cysteine side chains•Reaction can’t occur in the cytoplasmExample: antibodiesMany proteins are glycosylated in the ER1.Polypeptide is translocated into ER lumen2.Pre-formed, branched oligosaccharide (14 sugars) transferred en bloc from dolichol (a specialized lipid) to specific sequence3.Sequence contains asparagine - N-linked4.Reaction catalyzed by oligosaccharide protein transferase (active site oriented towards lumen)Many proteins are glycosylated in the ERSome proteins are retained in the ER•Proteins containing ER retention signal are held in the ER lumen (i.e. for disulfide bond formation, oligosaccharide transferases)•Proteins that are misfolded or fail to oligomerize are held for quality control by chaperoninsThe Golgi apparatus is made of stacked, flattened membranesProteins are further modified in the Golgi apparatus•Proteins enter the Golgi via vesicle fusion with the cis face and pass through the Golgi between successive stacks by transport vesicles•Oligosaccharide chains added in the ER are modified by enzymes in the Golgi - addition and removal of sugars to make complex oligosaccharides•Reactions are processive - early-acting enzymes in the cis compartments, late acting enzymes in the trans compartments2 pathways for exocytosis: regulated and constitutiveExocytosis occurs via 2 distinct pathways: regulated and constitutive•Constitutive exocytosis: A steady stream of delivery occurring in all cells. Plasma membrane components to replace endocytosed material and for membrane growth.•Regulated exocytosis: Operates only in cells specialized for secretion (i.e. secretory cells in the gut and glands). Secretory vesicles are docked at the plasma membrane until the cells receive an outside signal.Secretory vesicles package concentrated protein aggregates•Proteins in this pathway have special surface properties•The lumen of the TGN is acidic and has high calcium ==> aggregationThe endocytic pathway: •Two main mechanisms of endocytosis:•Phagocytosis: “cellular eating”; The ingestion of large particles (i.e. microorganisms, cellular debris) via large vesicles called phagosomes. Only occurs in specialized cells•Pinocytosis: “cellular drinking”; The ingestion of fluid and small molecules via small (<150 nm diameter) vesicles. Occurs in all cells.Phagocytic cells ingest large particlesQuickTime™ and aH.264 decompressorare needed to see this picture.Phagocytic cells ingest large particlesReceptor-mediated endocytosis is a specialized form of pinocytosis•Pinocytosis traps molecules in the extracellular fluid randomly•Receptor-mediated endocytosis traps specific molecules, concentrates them in vesicles•Both processes use clathrin-mediated vesicle formationEndosomes : “sorting stations” for membrane trafficking•Early endosomes (beneath the membrane) vs. late endosomes (in the center of the cell)•Lumen is kept acidic by ATP-driven proton pump - many (not all) ligands dissociate from their receptor•Receptors may be recycled, degraded, or undergo transcytosisExample: receptor-mediated endocytosis of LDLDifferent receptors have different fates•Some receptors are recycled to the same plasma membrane domain•Some are degraded in lysosomes•Some are recycled to a different domain (transcytosis)•Ligands that stay bound in endosome will follow their receptorIntracellular digestion occurs in lysosomes•Very acidic (to protect the cell)•~40 types of hydrolytic enzymes (acid hydrolases)•Resident proteins are protected by high levels of glycosylationAutophagy: a degradation pathway to dispose of obsolete parts of the cellThere are multiple pathways to the
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