3/1/15 1 Secretory Pathway Reading ECB3: 510-521 ECB4 503-515 Learning Objectives: • Understand the destinations of proteins synthesized on the ER • Understand that N-linked glycosylation occurs on proteins in the ER and is then modified in the Golgi • Explain what is meant by ER ‘quality control’ and how this is achieved in the ER • Understand the topology and mechanism of vesicular transport between membrane-bound compartments, including the roles of coat proteins, SNAREs and Rab proteins • Understand how the Golgi apparatus functions in protein sorting Review of targeting to the ER, SRP and translocation: Movie 15-4 Last Lecture……………3/1/15 2 One of the Greatest Cell Biologists: He discovered that secretory proteins are synthesized on the ER and transported through the Golgi to the plasma membrane George Palade 1912-2008 Nobel Prize 1974 Figure 15-18 Essential Cell Biology Roadmap of vesicle traffic Secretory pathway George Palade: discovered the secretory proteins are synthesized on the ER and transported through the Golgi to the plasma membrane3/1/15 3 The Endoplasmic Reticulum (ER) is a network of membranes spread throughout the cell. EM: Specialized secretory cell Fluorescence micrograph of ER at edge of cultured cell The ER is the entry point for membrane and lumenal proteins of the ER, Golgi, endosomes, lysosomes, and of secreted and plasma membrane proteins. (SRP and SRP receptor not shown) Golgi Apparatus Plasma membrane and secretory proteins Lysosomal Proteins Endosome3/1/15 4 Figure 15-18 Essential Cell Biology Roadmap of vesicle traffic Endocytic pathway Secretory pathway Today’s topics 1. Events in the ER: Protein glycosylation, protein folding, protein Quality Control, and the Unfolded Protein Response Pathway 2. Transport by vesicles to the Golgi Apparatus: COPII 3. Protein modification in the Golgi, and transport out of it. overall movement of vesicle traffic: ER -> golgi apparatus -> transport vesicles -> plasma membrane ER -> golgi apparatus -> early endosome -> plasma membrane plasma membrane -> early endosome -> late endosome -> lysosome- transport vesicles bud from 1 membrane and fuse with another - cytosolic side always faces cytosol - noncytosolic side faces the lumen of the compartment or the outside of the cell- extracellular space and each of the membrane-enclosed compartments communicate with one another by transport vesicles3/1/15 5 Many Proteins are Glycosylated on Asparagine in the ER: ‘N’-linked glycosylation Figure 15-23 Essential Cell Biology ……..Asn-X-Ser/Thr……..(where X is any amino acid except proline) (mRNA and Ribosome not shown) Called ‘N’-linked because N is the single letter code for asparagine NEVER occurs in the cytosol ONLY ever occurs in the ER lumen Chaperones assist protein folding and prevent misfolded proteins from leaving the ER Figure 15-24 Essential Cell Biology If the chaperone machinery cannot fold a protein properly, the protein is eventually degraded. Correct protein folding often involves disulfide bond formation between appropriate cysteine residues. This is called the ‘ER Quality Control System.’ Chaperone: a protein that helps other proteins to fold correctly. S-S S-S S-S -SH -SH - when appropriate asparagine enters the ER lumen, it is glycosylated by addition of a branched oligosaccharide side chain - each side chain is transferred from dolichol (lipid) to asparagineMisfolded proteins vs normally folded proteins: - misfolded: bind to chaperone proteins in ER lumen, stay in ER lumen - normal: transported in transport vesicles to the Golgi apparatusif misfolded proteins fail to refold normally they are transported back to the cytosol3/1/15 6 The ER adjusts its protein folding capacity when there are elevated levels of misfolded proteins: The Unfolded Protein Response (UPR) Figure 12-55a Molecular Biology of the Cell Figure 15-18 Essential Cell Biology Location of Cl- channel In unaffected individuals CFTR: Cystic Fibrosis Transmembrane Conductance Regulator – it is a Cl- channel normally found in the plasma membrane - accumulation of misfolded proteins in the ER lumen triggers unfolded protein response (UPR) Unfolded protein response (UPR) - misfolded proteins are recognized by several types of transmembrane sensor proteins in the ER membrane, each of which activates a different part of the UPR - allows cell to adjust the size of its ER according to the load of proteins entering the secretory pathway - if ER reaches max capacity, UPR can direct cell to self destruction by undergoing apoptosis activated sensorsactivated transcription regulators-Cystic Fibrosis: leads to severe lung damage. Produces a plasma membrane transport protein that is misfolded. Protein is retained in the ER. Protein could function normally as a chloride channel but isn't given the opportunity. causes the retention and degradation of the CFTR protein in the ER by the Quality Control System3/1/15 7 The disease cystic fibrosis is due to a mutation in the gene for CFTR that causes the retention and degradation of the CFTR protein in the ER by the Quality Control System Mutant CFTR does not leave the ER and is degraded by the Quality Control System Today’s topics 1. Events in the ER: Protein glycosylation, protein folding, protein Quality Control, and the Unfolded Protein Response Pathway 2. Transport by vesicles to the Golgi Apparatus: COPII 3. Protein modification in the Golgi, and transport out of it.3/1/15 8 Nucleus The Secretory Pathway and Protein Sorting ER ‘resident’ protein Protein to be secreted out of cell Protein to be delivered to the lysosome Selective transport occurs by vesicles Figure 13-25b Molecular Biology of the Cell (© Garland Science 2008) 1µm Nuclear envelope Rough ER Golgi Transport vesicles Transport vesicles3/1/15 9 Figure 13-2 Molecular Biology of the Cell Vesicle budding and fusion: General Principles The asymmetry of the lipid bilayer is preserved, as is the orientation of membrane proteins The topology of the lipid bilayer and membrane proteins is preserved Figure 13-2 Molecular Biology of the Cell Vesicle budding and fusion with the plasma membrane The topology of the lipid bilayer and membrane proteins is preserved - vesicles that bud from the membranes usually have a distinctive protein coat on their cytosolic surface -> called coated vesicles - after budding from its parent organelle,