Trafficking 1 MCB 110 - Spring 2008- Nogales 4 MEMBRANE TRAFFICKING I Introduction: secretory pathway A. Protein Synthesis and sorting B. Methods to study cytomembranes II Endoplasmic Reticulum A. Smooth ER B. Rough ER C. Synthesis of proteins in membrane-bound ribosomes • The signal hypothesis • Synthesis of membrane proteins D. Glycosylation in the ER III Golgi IV Vesicle Transport A. COPII-coated Vesicles B. COPI-coated Vesoicles C. Clathrin-coated Vesicles V Lisosomes A. Phagocytosis B. Autophagy VI Endocytosis Suggested Reading: Lodish, Chapter 5, 5.3; Chapter 16, 16.1 to 16.3; Chapter 17 Alberts, Chapters 12 and 13 Trafficking 2 MCB 110 - Spring 2008- Nogales I Introduction to the Secretory Pathway The eukaryotic cell is filled with membranous organelles that form part of an integrated and dynamic system shuttling material across the cell Trafficking 3 MCB 110 - Spring 2008- Nogales The biosynthetic or secretory pathway includes the synthesis of proteins in the ER, their modification in the ER and Golgi, and their transport to different destinations such as the plasma membrane, lysosomes, vacuoles, etc. In constitutive secretion materials are transported in a continual manner. In regulated secretion , materials are stored in secretory granules in the periphery of the cell and discarded in response to a particular stimulus (e.g. nerve cells, cells producing hormones or digestive enzymes). Materials are transported in vesicles that move along microtubules, powered by motor proteins. Sorting is facilitated by receptors localized in particular membranes. Trafficking 4 MCB 110 - Spring 2008- Nogales Methods to Study Cytomembranes Visualization by electron microscopy Dynamic localization by autoradiography and pulse-chase Trafficking 5 MCB 110 - Spring 2008- Nogales Trafficking 6 MCB 110 - Spring 2008- Nogales Use of GFP constructs Movement of proteins through the secretory pathway has been followed using a Green Flourescence Protein (GFP). Cells where infected with vesicular stomatitis virus (VSV) in which one of the genes (VSVG) is fused to GFP. Large amounts of VSVG protein are produced in the ER that move to the Golgi and then to the plasma membrane. The process can be seen as a wave of green fluorescence that can be synchronized using temperature mutants of VSVG than cannot leave the ER at high temperatures. Trafficking 7 MCB 110 - Spring 2008- Nogales Subcellular fraction purification and characterization: Differential Centrifugation & Cell Fractionation. When cells are homogenized the rough ER breaks up into small closed vesicles call microsomes. Trafficking 8 MCB 110 - Spring 2008- Nogales Cell-Free Systems Inmediately after their synthesis, secretory proteins are localized in the lumen of microsomes. If proteases are added to the microsomes, the secretory protein is not digested. If the microsomes are treated with detergent previous to their exposure to proteases, the secretory protein is digested. Trafficking 9 MCB 110 - Spring 2008- Nogales Mutant 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 secretion of proteins is block at the non-permissive temperature. These are called sec mutants. The analysis of these mutants identified 5 classes (A-E) that correspond to 5 steps in the secretory pathway with distinctive distribution of cytoplasmic membranes. Trafficking 10 MCB 110 - Spring 2008- Nogales Trafficking 11 MCB 110 - Spring 2008- Nogales Protein Targeting Trafficking 12 MCB 110 - Spring 2008- Nogales Trafficking 13 MCB 110 - Spring 2008- Nogales Trafficking 14 MCB 110 - Spring 2008- Nogales Endoplasmic Reticulum Smooth ER Lacks ribosomes Tubular Membranes Functions: M Synthesis of steroid hormones Detoxification of the liver Secuestration of Ca2+ (skeletal muscle) SER RER Rough ER Ribosomes on the cytosolic side Flattened stacks Functions: Synthesis of secretory proteins Granule • Intestinal cells: mucoproteins • Endocrine cells: polypeptide hormones • Plasma cells: antibodies • Liver cells: blood serum proteins Synthesis of membrane proteins Synthesis of soluble, endomembrane proteins Post-translational modification Protein folding/control Trafficking 15 MCB 110 - Spring 2008- Nogales Trafficking 16 MCB 110 - Spring 2008- Nogales Synthesis of Proteins on Membrane-bound Ribosomes The Signal Sequence Hypothesis Trafficking 17 MCB 110 - Spring 2008- Nogales Trafficking 18 MCB 110 - Spring 2008- Nogales Electron Microscopy and 3-D Reconstruction General Applicability 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 Trafficking 19 MCB 110 - Spring 2008- Nogales Imaging and Reconstruction of Biological Macromolecules EM + Noise 3D 2D Averaging Volume (3D) Projection (2D) Experimental Image Trafficking 20 MCB 110 - Spring 2008- Nogales SRP-Ribosome Structure Trafficking 21 MCB 110 - Spring 2008- Nogales Experimental prove of the cotranslational insertion of secretory proteins into the ER. Trafficking 22 MCB 110 - Spring 2008- Nogales Experimental identification of proteins that form the translocon. Trafficking 23 MCB 110 - Spring 2008- Nogales Trafficking 24 MCB 110 - Spring 2008- Nogales Translocon-Ribosome Structure Trafficking 25 MCB 110 - Spring 2008- Nogales Trafficking 26 MCB 110 - Spring 2008- Nogales Synthesis of Integral Membrane Proteins Trafficking 27 MCB 110 - Spring 2008- Nogales Trafficking 28 MCB 110 - Spring 2008- Nogales Trafficking 29 MCB 110 - Spring 2008- Nogales Trafficking 30 MCB 110 - Spring 2008- Nogales Post-translational modifications and quality control in the RER Both the soluble and membrane proteins synthesized in the ER undergo several modifications 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 redox environment 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 multimeric proteins is facilitated by several ER proteins. Only those proteins that are properly folded can progress to the Golgi, those misfolded or unassembled are transported to the cytosol where they are degraded. Addition and processing of carbohydrates – Glycosylation of many plasma-membrane and secretory proteins is initiated in the ER and continues in the Golgi. Trafficking 31 MCB 110 - Spring 2008- Nogales II D – Glycosylation in the ER • Most proteins synthesized in the ER become glycosylated. • The sequence of sugars in the oligosaccharide are highly specific •Carbohydrates are important for interaction with other macromolecules. They also confer extra stability to many extracellular glycoproteins. • Addition of sugars is catalyzed by membrane-bound enzymes called glycosyltransferases. Oligosaccharides can be added to a protein through an asparagine (N- linked). This occurs both in the ER and the Golgi. N-linked oligosaccharides tend to be large and branched. Synthesis starts by the addition of a large precursor to the protein. They can also be added via a serine or a threonine (O-linked). This occurs only in the Golgi. O-linked oligosaccharides tend to be short, one to four sugars in length. Sugars are added one at a time. Trafficking 32 MCB 110 - Spring 2008- Nogales Production of N-linked Oligosaccharides •The core of the oligosaccharide is assembled on a carrier molecule, dolichol phosphate. • 1-9: sugars are added one at a time, with the first steps taking place in the cytosol and the rest in the ER cisternae. The donor is always a nucleotide: CMP-sialic acid GDP-mannose UDP-N-acetylglucosamine 10: A finally assembled block of 14 sugars is transferred by oligosaccharidetransferase to an asparagine residue in the protein as is being translocated into the ER. 11-13: Dolichol phosphate is recycled for a new round of assembly Modification of this core oligosaccharide starts in the ER with the removal of the terminal glucose residues by glucosidases. Trafficking 33 MCB 110 - Spring 2008- Nogales Trafficking 34 MCB 110 - Spring 2008- Nogales III Golgi Formed by flattened, disk-like cisternae with dilated rims, associated tubules and vesicles. Functional compartments: • Cis Golgi network (CGN) - Sorting of proteins between ER and Golgi. • Cis, medial, trans cisternae - sequential protein modification: • proteolytic cleavage • amino acid modifications • Carbohydrates modifications • Trans Golgi network (TGN) - Sorting of vesicles to different destinations Trafficking 35 MCB 110 - Spring 2008- Nogales Differential staining of Golgi compartments Osmium tetraoxide in Antibodies for Mannosidase II in Antibodies for diphosphatase cis cisternae Medial cisternae in Trans cisternae Trafficking 36 MCB 110 - Spring 2008- Nogales Models of Transport within the Golgi There are two opposing models on how materials move through the Golgi system. • In the maturation model each cis cisterna matures into a transcisterna. The evidence comes from cells that produce secretory products too large to fit into vesicles. • In the alternative model cisternae stay in place and movement occurs through vesicles budding from one cisterna and fusing into the next. The evidence comes from reconstituted vesicle transport