March 30, 2006Pabio552, Lecture 21 For anyone having trouble understanding retrotranslocation, quality control in the ER, and molecular chaperones (from lecture 1), here are two reviews that should help: Spiess C, Meyer AS, Reissmann S, Frydman J. Mechanism of the eukaryotic chaperonin: protein folding in the chamber of secrets. Trends Cell Biol Nov;14(11): Review. Tsai B, Ye Y, Rapoport TA.Tsai B, Ye Y, Rapoport TA. Retro-translocation of proteins from the endoplasmic reticulum into the cytosol. Nat Rev Mol Cell Biol Apr;3(4): Review.

March 30, 2006Pabio552, Lecture 22 Lecture 2: Trafficking from ER to Golgi: Outline: A.Important concepts in trafficking: 1.ER as the gateway to the secretory pathway 2.ER translocation is co-translational 3.Signals direct translocation across membranes 4.Conservation of topology 5.Molecular sorting keeps membranes biochemically distinct 6.Vesicle targeting involves specific address tags B.Specific events in vesicular traffic 1.Definitions 2.Exocytosis 3.Lysosomal Sorting 4.Endocytosis 5.Vesicle fusion (covered in PM lecture)

March 30, 2006Pabio552, Lecture 23 Trafficking from ER to Golgi: Outline, cont.: C. Machinery and Mechanisms of Trafficking 1. Overview 2. Types of coats: Clathrin, Cop I, Cop II 3. Proteins that function with coats D. Experimental Systems 1. Cell-free reconstitution of Golgi transport 2. Biochemical analysis of synaptic vesicle membranes 3. Genetic dissection of yeast secretion 4. EM and Fluorescence microscopy E. Examples from Pathobiology How viruses exploit trafficking pathways The lysosomal dogma turned on its head

March 30, 2006Pabio552, Lecture 24 Trafficking : ER to Golgi to Lysosome A.Important Concepts in Trafficking: 1. The ER as the secretory pathway gateway. Entry into the ER allows trafficking to specific compartments including ER, Golgi, endosomes, lysosomes, plasma membrane and cell exterior. 2. ER translocation is co-translational in higher eukaryotic cells. In contrast, import into ER of yeast (lower eukaryotes) is post-translational. In addition, import into nucleus, mitochondria, and peroxisomes is post- translational. Note that flow in the secretory pathway goes in multiple directions. The Cell: A Molecular Approach, 3 rd Ed (Cooper). ASM Press, 2000

March 30, 2006Pabio552, Lecture 25 A 2. ER translocation is co-translational

March 30, 2006Pabio552, Lecture 26 Please review the signal hypothesis and mechanisms of co-translational translocation in the Alberts’ textbook!

March 30, 2006Pabio552, Lecture 27 Please review the signal hypothesis and mechanisms of co-translational translocation in the Alberts’ textbook!

March 30, 2006Pabio552, Lecture 28 Trafficking : ER to Golgi to Lysosome A. Important Concepts in Trafficking: 3. Signals direct translocation across membranes. ”N-terminal signal sequences”: the cannonical example of signals that mediate trafficking. Entry into the secretory pathway requires a signal sequence (SS). SS overcome unfavorable energetics in protein transfer across membranes. Variations in SS: stop-transfer sequences (signal anchor) & internal signals. At the ER: SS allow interaction of nascent chain with SRP & SRP receptor, leading to cotranslational translocation through the translocon. Variations in SS directed translocation occurs in bacteria, yeast, eukaryotes, & in different compartments (i.e. co-translational vs. post- translational, translocation into other organelles). Other types of signals are important in trafficking besides SS - to be covered in upcoming lectures.

March 30, 2006Pabio552, Lecture 29 We’ll fill these lanes in during class

March 30, 2006Pabio552, Lecture 210 Trafficking : ER to Golgi to Lysosome A. Important Concepts in Trafficking: 4. Conservation of topology: i. Membrane sidedness (lumenal vs. cytoplasmic) is maintained throughout the secretory pathway: Why? Because there is lateral mobility of lipid & protein in lipid bilayers, but typically no spontaneous flip-flop across bilayers (this is energetically unfavorable).

March 30, 2006Pabio552, Lecture 211 Trafficking : ER to Golgi to Lysosome A.Important Concepts in Trafficking: 4. Conservation of topology: ii. Lumen = lumen = extracellular space Why is this? It follows from how the ER evolved in primitive eukaryotes. Evolution of the Eukaryotic ER

March 30, 2006Pabio552, Lecture 212 Trafficking : ER to Golgi to Lysosome A.Important Concepts in Trafficking: 4. Conservation of topology: ii. Lumen = lumen = extracellular space

March 30, 2006Pabio552, Lecture 213 Trafficking : ER to Golgi to Lysosome A.Important Concepts in Trafficking: 4. Conservation of topology:

March 30, 2006Pabio552, Lecture 214 Trafficking : ER to Golgi to Lysosome A.Important Concepts in Trafficking: 4. Conservation of topology:

March 30, 2006Pabio552, Lecture 215 Trafficking : ER to Golgi to Lysosome A.Important Concepts in Trafficking: 5. Molecular sorting keeps membranes in each compartment "biochemically distinct" despite continuous vesicular traffic between compartments. Involves balance of forward and backwards transport. Types of sorting: A. Selection of specific components during formation of TV B. Segregation of vesicular container from cargo after fusion. C. Retrieval of specific components for retrograde transport. i.e. proteins bearing KDEL and KKXX sequences bind to specific recycling receptors in the Golgi and are selectively transported back to the ER.

March 30, 2006Pabio552, Lecture 216 Trafficking : ER to Golgi to Lysosome A.Important Concepts in Trafficking: 6. Vesicle targeting involves specific address tags that identify donor vesicle and target organelle. examples: V-SNARES vs. T-SNARES, Rab GTPases that are specific for different organelles or vesicles. The Cell: A Molecular Approach, 3 rd Ed (Cooper). ASM Press, 2000

March 30, 2006Pabio552, Lecture 217 Trafficking : ER to Golgi to Lysosome B. Specific Events in Vesicular Trafficking: 1. Definitions: Exocytosis - fusion of vesicles derived from TGN with the PM resulting in insertion of transmembrane proteins into PM or secretion of soluble proteins into extracellular space. Endocytosis - process by which particles, solutes, membrane proteins (including receptor-ligand complexes) and lipids are taken up by vesicles from the PM. Also used by parasites and bacteria to get into the host cell (to be discussed in Nancy Freitag's upcoming lecture). Phagocytosis - uptake of pathogens as a defense, clearance of cell debris. Pinocytosis - uptake of extracellular fluid through endocytosis.

March 30, 2006Pabio552, Lecture 218 Trafficking : ER to Golgi to Lysosome B.Specific Events in Vesicular Trafficking 2. Exocytosis a. Events that occur in the ER (review): Translocation, signal cleavage, N-linked core glycosylation Proper folding by ER resident chaperones Post-translational modifications: trimming core sugars, adding GPI anchors Retention of ER proteins via KDEL sequences b. Trafficking from ER to Golgi complex: Transport vesicles (TV): bud from mb of one organelle & fuse with mb of next organelle Golgi complex: a series of stacked membranes where proteins from ER are further processed (i.e. glycosylation, trimming, and CHO addition), & sorted for transport to final destinations: outside cell, PM, or lysosomes Consists of cis Golgi, Golgi stack (medial and trans), and trans Golgi network (TGN) Distinct polarity of Golgi: entry via the cis face; exit from the trans face.

March 30, 2006Pabio552, Lecture 219 The Cell: A Molecular Approach, 3 rd Ed (Cooper). ASM Press, 2000

March 30, 2006Pabio552, Lecture 220 Trafficking : ER to Golgi to Lysosome B.Specific Event, Vesicular Trafficking 3. Lysosomal sorting A.Lysosomes: v Organelle containing enzymes that degrade proteins, nucleic acids, CHO and lipids. v Delivery from exterior (via receptor-mediated endocytosis or phagocytosis) or from ER via Golgi. Lysosomal enzymes: v Acid hydrolases (i.e. cathepsin D), v Active at pH 5 (pH maintained within lysosomes) v Inactive at neutral pH. Protects cell in case of release into the neutral cytoplasm. v Acid pH maintained by ATP-dependent H+ pump in membrane. B. Trafficking of lysosomal enzymes: sugar N oligo-saccharide core added in the ER to lysosomal enzymes. 2. One mannose and 3 glucoses are removed while protein is still in the ER. The Lysosome J. R. Lingappa, Pabio 552, Lecture 2-14

March 30, 2006Pabio552, Lecture 221 Trafficking : ER to Golgi to Lysosome 3. Lysosomal sorting B. Trafficking of lysosomal enzymes, cont.: 3. Mannose residues on lysosomal enzymes are phosphorylated creating M-6-P residues. enzyme: N-acetyl-glucosamine (GlcNAc) phosph-transferase, which recognizes a specific conformation (signal patch) present only on lys. enzymes. site: cis Golgi. 4. Another enzyme removes GlcNac leaving M-6-P residue on lysosomal enzyme. The Cell: A Molecular Approach, 3 rd Ed (Cooper). ASM Press, 2000

March 30, 2006Pabio552, Lecture 222 Trafficking : ER to Golgi to Lysosome 4.Lysosomal sorting, cont. B. Trafficking of lysosomal enzymes, cont.: 5. The M-6-P residue binds to M-6-P receptors located in trans Golgi. Binding occurs at pH = (pH of Golgi), but not at pH < Clathrin-coated vesicles bud from trans Golgi, become uncoated, & fuse with late endosome. The Cell: A Molecular Approach, 3 rd Ed (Cooper). ASM Press, 2000

March 30, 2006Pabio552, Lecture 223 Trafficking : ER to Golgi to Lysosome 4.Lysosomal sorting, cont. B. Trafficking of lysosomal enzymes, cont.: 7.Late endosome (LE) pH = 5.5 so lyso enzyme is released from M-6-P receptor. Phosphatase in LE removes phosphate to prevent rebinding. 8.Transport vesicles (TV) transport enzymes to lysosomes 9.Some lysosomal enzymes need to undergo proteolytic cleavage (in lysosome) to become active 10.A different TV recycles M-6-P receptor back to trans-Golgi The Cell: A Molecular Approach, 3 rd Ed (Cooper). ASM Press, 2000

March 30, 2006Pabio552, Lecture 224 Trafficking : ER to Golgi to Lysosome 4. Lysosomal sorting C.Trafficking PM to lysosome: 1.Receptors take up cargo via receptor-mediated endocytosis (next lecture), into clathrin-coated endocytic vesicles which bud into cell from PM and fuse with early endosomes. 2.Early endosomes mature into late endosomes, with recycling of membrane components back to PM. 3.Late endosomes mature into lysosomes, resulting in lowering pH to 5.5, allowing the lysosomal enzyme activation. Note: M-6-P receptor is also transported to the PM where it binds extracellular phosphorylated lysosomal enzymes that are occasionally secreted. Thus proteins that are accidentally sent to one compartment can be rescued and brought back to another compartment.

March 30, 2006Pabio552, Lecture 225 Overview of Trafficking to the Lysosome The Cell: A Molecular Approach, 3 rd Ed (Cooper). ASM Press, 2000

March 30, 2006Pabio552, Lecture 226

March 30, 2006Pabio552, Lecture 227 J. R. Lingappa, Pabio 552, Lecture 2-21 Clathrin-Coated Vesicles: Trafficking: ER to Golgi to Lysosome C.Machinery and Mechanisms of Trafficking 2.Types of coats: a. Clathrin Acts during uptake of extracellular molecules at PM in endoctyosis Acts during lysosomal sorting in the TGN Structure: three-legged trimer of 3 HC and 3 LC Oligomerizes to form polyhedral lattice in coated pit Undergoes rearrangement to form curvature that results in budding Assembly/disassembly regulated by Hsp70 using ATP hydrolysis Clathrin Structure

March 30, 2006Pabio552, Lecture 228 Different Coat Proteins Act at Specific Points in the Secretory Pathway

March 30, 2006Pabio552, Lecture 229 J. R. Lingappa, Pabio 552, Lecture 2-22 Trafficking: ER to Golgi to Lysosome C.Machinery and Mechanisms of Trafficking 2. Types of Coats : b. CopI: Made of coatamer subunits. Mediates retrieval of proteins from Golgi to ER (retrograde transport). COPI vesicles transport ER resident proteins with KKXX or RRXX signals. Uses GTP binding protein ARF (as does clathrin). Note: The drug Brefeldin A inhibits activation of the ARF protein by inhibiting nucleotide exchange, and thereby inhibits budding of COPI vesicles. c. CopII: Mediates forward movement of vesicles from ER to Golgi (anterograde transport). Regulated by a GTP binding protein Sar1. Budding of COPII is not inhibited by Brefeldin A (which is specific for Arf).

March 30, 2006Pabio552, Lecture 230 Trafficking : ER to Golgi to Lysosome C. Cellular Machinery, Coats and Adaptors: 3. Proteins that function with clathrin: i. Adaptor proteins promote clathrin assembly, linking clathrin to the membrane and interacting with membrane proteins encoding signals for sorting into CCVs. ii. GTP-binding proteins (include ARF and Sar1) regulate coat protein binding: Sar1 or ARF bound to GTP recruits coat proteins to vesicle. Coat proteins promote bud formation. After budding occurs, GTP is hydrolyzed to GDP resulting in dissociation of coat proteins from vesicle. Guanine Exchange Factors (GEFs) exchange GDP and replace with GTP

March 30, 2006Pabio552, Lecture 231 Model for Dynamin Action Vesicles in Shibire mutant Trafficking: ER to Golgi to Lysosome C.Machinery and Mechanisms of Trafficking 3. Proteins that function with coats: c. Dynamin, a GTPase protein: Localizes to membrane-bud junction to cause vesicle closure (coated pit becomes vesicle). Purified dynamin can constrict vesicles to form long tubelike structures. Dynamin activity is probably regulated by a kinase-phosphatase cycle. Other proteins (i.e. amphiphysin) implicated dynamin recruitment from cytosol. Temperature-sensitive dynamin mutants in drosophila (shibire) undergo paralysis due to accumulation of long-neck coated pits and failure to generate coated vesicles in neurosecretory cells.

March 30, 2006Pabio552, Lecture 232 Trafficking: ER to Golgi to Lysosome C.Machinery 4. Summary The Cell: A Molecular Approach, 3 rd Ed (Cooper). ASM Press, 2000

March 30, 2006Pabio552, Lecture 233 Example of Genetics Used For Studying Trafficking Trafficking : ER to Golgi to Lysosome D. Experimental systems used to study trafficking: 1. Cell-free reconstitution of Golgi transport (i.e. Rothman & colleagues) Uses cytoplasmic extracts, ER plus Golgi membranes, and de novo synthesis of radiolabeled proteins off mRNA transcripts to study protein trafficking. Particularly useful for identifying novel cellular machinery. 2. Biochemical analysis of different membranes. 3. Genetic dissection of yeast secretion (i.e. Schekman & colleagues). Sec mutants = yeast mutants defective in various stages of vesicular transport, i.e. protein secretion, vacuolar transport, or retrieval of ER resident proteins. Isolation of mutants led to molecular cloning of genes. 4. Electron microscopy; Fluorescence microscopy Molecular Biology of the Cell, 4th edition

March 30, 2006Pabio552, Lecture 234 Cell-free systems for studying trafficking Molecular Biology of the Cell, 4th edition

March 30, 2006Pabio552, Lecture 235 Trafficking: ER to Golgi to Lysosome E. Examples from Pathobiology:

March 30, 2006Pabio552, Lecture 236

March 30, 2006Pabio552, Lecture 237 Additional Reading on Pathogens and Intracellular Trafficking Pathways Overviews: Knodler, L. A., J. Celli, and B. B. Finlay. Pathogenic Trickery: deception of host cell processes. Nature Rev. Mol. Cell. Bio. 2: (2001)(review) Reprogramming the phagocytic pathway - intracellular pathogens and their vacuoles. Mol. Memb. Biol. 15: (1998) Specific Organisms: Stegmann, T. Membrane fusion mechanisms: The influenza hemagglytinin paradigm and its implications for intracellular fusion. Traffic 1:598 (2000). Portnoy, D. A., V. Auerbuch, and I. J. Glomski. The cell biology of Listeria monocytogenes infection: the intersection of bacterial pathogenesis and cell-mediated immunity. J. Cell Bio 158: (2002) (minireview) Roy, C. R. and L. G. Tilney. The road less traveled: transport of Legionella to the endoplasmic reticulum. J. Cell Bio 158: (2002) (minireview) Russell, D. G., H. C. Mwandumba, and E. E. Rhoades. Mycobacterium and the coat of many lipids. J. Cell Bio 158: (2002) (minireview) Andrews, N. W. Lysosomes and the plasma membrane: trypanosomes reveal a secret relationship. J. Cell Bio 158: (2002) (minireview)