MCB 110 - Spring 2008- NogalesTrafficking 14 MEMBRANE TRAFFICKINGI Introduction: secretory pathwayA. Protein Synthesis and sortingB. Methods to study cytomembranesII Endoplasmic ReticulumA. Smooth ERB. Rough ERC. Synthesis of proteins in membrane-bound ribosomes• The signal hypothesis• Synthesis of membrane proteinsD. Glycosylation in the ERIII GolgiIV Vesicle TransportA. COPII-coated VesiclesB. COPI-coated VesoiclesC. Clathrin-coated VesiclesV LisosomesA. PhagocytosisB. AutophagyVI EndocytosisSuggested Reading: Lodish, Chapter 5, 5.3; Chapter 16, 16.1 to 16.3; Chapter 17Alberts, Chapters 12 and 13MCB 110 - Spring 2008- NogalesTrafficking 2I Introduction to the Secretory PathwayThe eukaryotic cell is filled with membranous organelles that form part ofan integrated and dynamic system shuttling material across the cellMCB 110 - Spring 2008- NogalesTrafficking 3The biosynthetic or secretory pathwayincludes the synthesis of proteins in the ER,their modification in the ER and Golgi, andtheir transport to different destinationssuch as the plasma membrane, lysosomes,vacuoles, etc.In constitutive secretion materials aretransported in a continual manner.In regulated secretion , materials are storedin secretory granules in the periphery of thecell and discarded in response to a particularstimulus (e.g. nerve cells, cells producinghormones or digestive enzymes).Materials are transported in vesicles thatmove along microtubules, powered by motorproteins. Sorting is facilitated by receptorslocalized in particular membranes.MCB 110 - Spring 2008- NogalesTrafficking 4Methods to Study CytomembranesVisualization by electron microscopyDynamic localization by autoradiography and pulse-chaseMCB 110 - Spring 2008- NogalesTrafficking 5MCB 110 - Spring 2008- NogalesTrafficking 6Use of GFP constructsMovement of proteins through the secretory pathway has been followed using aGreen Flourescence Protein (GFP). Cells where infected with vesicular stomatitisvirus (VSV) in which one of the genes (VSVG) is fused to GFP. Large amounts ofVSVG protein are produced in the ER that move to the Golgi and then to theplasma membrane. The process can be seen as a wave of green fluorescence thatcan be synchronized using temperature mutants of VSVG than cannot leave the ERat high temperatures.MCB 110 - Spring 2008- NogalesTrafficking 7Subcellular fraction purification and characterization:Differential Centrifugation & Cell Fractionation.When cells are homogenized the rough ER breaks up intosmall closed vesicles call microsomes.MCB 110 - Spring 2008- NogalesTrafficking 8Inmediately after their synthesis,secretory proteins are localized inthe lumen of microsomes.If proteases are added to themicrosomes, the secretory proteinis not digested.If the microsomes are treatedwith detergent previous to theirexposure to proteases, thesecretory protein is digested.Cell-Free SystemsMCB 110 - Spring 2008- NogalesTrafficking 9Mutant Studies Yeast cells: they are small, fast growing and haploid Yeast continuously secrete a number of proteins, one of them is invertase.Temperature-sensitive mutants strains have been identified where the secretionof proteins is block at the non-permissive temperature. These are called secmutants.The analysis of these mutants identified 5 classes (A-E) that correspond to 5 stepsin the secretory pathway with distinctive distribution of cytoplasmic membranes.MCB 110 - Spring 2008- NogalesTrafficking 10MCB 110 - Spring 2008- NogalesTrafficking 11Protein TargetingMCB 110 - Spring 2008- NogalesTrafficking 12MCB 110 - Spring 2008- NogalesTrafficking 13MCB 110 - Spring 2008- NogalesTrafficking 14Endoplasmic ReticulumSmooth ERLacks ribosomesTubular MembranesFunctions:Synthesis of steroid hormonesDetoxification of the liverSecuestration of Ca2+ (skeletal muscle)Rough ERRibosomes on the cytosolic sideFlattened stacksFunctions:Synthesis of secretory proteins• Intestinal cells: mucoproteins• Endocrine cells: polypeptide hormones• Plasma cells: antibodies• Liver cells: blood serum proteinsSynthesis of membrane proteinsSynthesis of soluble, endomembrane proteinsPost-translational modificationProtein folding/controlMSERRERGranuleMCB 110 - Spring 2008- NogalesTrafficking 15MCB 110 - Spring 2008- NogalesTrafficking 16Synthesis of Proteins on Membrane-bound RibosomesThe Signal Sequence HypothesisMCB 110 - Spring 2008- NogalesTrafficking 17MCB 110 - Spring 2008- NogalesTrafficking 18Electron Microscopy and 3-D Reconstruction No crystallization is required Applicable to very large complexes Requires very small amounts of sampleStudy of Fully Assembled, Functional Complexes- In near physiological conditions- In different functional states- Structural Basis of Function and RegulationGeneral ApplicabilityMCB 110 - Spring 2008- NogalesTrafficking 19Projection (2D)EMVolume (3D)+ NoiseExperimentalImageAveraging3D 2DImaging and Reconstruction of Biological MacromoleculesMCB 110 - Spring 2008- NogalesTrafficking 20SRP-Ribosome StructureMCB 110 - Spring 2008- NogalesTrafficking 21Experimental prove of the cotranslational insertion of secretory proteinsinto the ER.MCB 110 - Spring 2008- NogalesTrafficking 22Experimental identification of proteins that form the translocon.MCB 110 - Spring 2008- NogalesTrafficking 23MCB 110 - Spring 2008- NogalesTrafficking 24Translocon-Ribosome StructureMCB 110 - Spring 2008- NogalesTrafficking 25MCB 110 - Spring 2008- NogalesTrafficking 26Synthesis of Integral Membrane ProteinsMCB 110 - Spring 2008- NogalesTrafficking 27MCB 110 - Spring 2008- NogalesTrafficking 28MCB 110 - Spring 2008- NogalesTrafficking 29MCB 110 - Spring 2008- NogalesTrafficking 30Post-translational modifications and quality control in the RERBoth the soluble and membrane proteins synthesized in the ER undergo severalmodifications before they move to Golgi:Formation of disulfide bonds – This modification can occur in the ER but not in the cytosol.Although the enzyme that catalyzes the reaction has not been identified, the redoxenvironment in the ER is more appropriate for the oxidation of sulphydryl groups (-SH).Folding – The proper folding of new proteins and the assembly of subunits into multimericproteins is facilitated by several ER proteins. Only those proteins that are properly foldedcan progress to the Golgi, those misfolded or unassembled are transported to the cytosolwhere they are degraded. Addition and processing of carbohydrates – Glycosylation