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Lecture 16 Vesicle transport and targeting in the secretory pathway COP coated vesicles SNAREs Protein sorting Secretion - Golgi to plasma membrane Retention in ER Golgi to lysosome Transport between organelles is mediated by coated vesicles Clathrin coated vesicles mainly involved in endocytosis (next time) COP coated vesicles mediate ER to Golgi and back Transport between ER and Golgi compartments occurs via “COP-coated vesicles”… Collection of 4-7 “coat proteins” = “COPs”…(aka “Coatomers” ) COP-coated vesicles function in transport between: ER and Golgi Golgi and ER (retrieval) intra-Golgi TGN and plasma membrane Cop coated vesicles contain many proteins COP proteins More COP proteins “cargo” Lipid bilayer Sar1 COPII-coated vesicles - ER to GolgiSarI in ER membrane COPI coated vesicles - Golgi to ER ARF (instead of Sar1) in Golgi membrane We will only consider Sar1 Sar1 ARF triggers vesicle formation Sar1: GTPase switch on/off ON: binds membrane recruits COP proteins COP proteins then recruit specific cargo Sar1 -Similar to RAN in nuclear import GTPase (GTP Binding Proteins) Large family (Ras) of proteins Molecular “switches” Pi GAP GDP GTP Sar1 GTPase “on” Bound to membrane In cytoplasm, large amount in “off” form GTP GEF Sar1 GTPase “off” GDP cytoplasmic Sar1 activation exposes hydrophobic tail and membrane insertion Greasy foot Sar 1 in membrane recruits COP proteins The Ras “superfamily” of small GTPases… • Ras: signaling and regulating cell proliferation… >30% of human tumors have Ras mutations… Many (not all) Ras family members associated with membranes via covalent fatty acid tail (“greasy feet”)… • EF-1/EF-Tu: translation… • Ran: nuclear transport… • Rho family (Rho, Rac, cdc42): actin assembly and organization (in a few lectures) • Arf/Sar family of “Coat recruitment GTPases:” COP assembly and vesicle budding… • Rab family: vesicle targeting and fusion (in a few minutes) Aside: G-proteins and ATPases as molecular switches Cells make high-affinity transient molecular complexes as trigger or switch A B Bound GTP GDP + Pi A paradox: High-affinity/high-specificity = stable… A + B Unbound Energy input is required to dissociate high-affinity complexes… (Example: to remove Sar 1 from membrane) Translation: IFs (GTP), EF-1/EF-Tu (GTP) EF-2/EF-G (GTP) Chaperones: HSP70 family (ATP) HSP60 (ATP) SRP family: SRP54 (GTP), SRP-Ra (GTP) SRP-Rb (GTP) Polymer dynamics: Actin (ATP), Tubulin (GTP) Dynamin (GTP) Motors: Myosin (ATP), Dynein (ATP) Kinesin (ATP) Signaling: Heterotrimeric G proteins (GTP) Ras family (GTP) Summary of COPII-coated vesicle formation COP subunits recruit specific cargo proteins… Vesicle transport is a complex process 2. Formation of coated transport vesicle… 3. Targeting and docking to specific compartment… SNAREs and Rabs Target compartment 1. Formation of coated buds… (ATP, GTP, and cytoplasmic protein factors…) Donor compartment Coat proteins (“COPs”) The Snare hypothesis: v- and t-SNAREs target transport vesicles to the correct membrane Budding Cargo v-SNAREs Uncoating, targeting and docking t-SNAREs See ECB figure 15-20 Specific pairing of V-SNAREs with T-SNAREs matches vesicle to target membrane compartment (>20 known snares in animals cells) Targeting and docking requires/is facilitated by specific Rab GTPase in vesicle and Rab effector in target (~30 known Rabs in animal cells)… Bacterial toxins target the vesicle docking and fusion machinery of neurons A small subunit of the toxin acts as a specific protease that cleaves and inactivates targeting proteins Botulism A SNAP25 (t-SNARE) Botulism B VAMP (v-SNARE) Botulism C Syntaxin (t-SNARE) Tetanus VAMP (v-SNARE) Net result is to block neuronal signaling by blocking neurotransmitter release (regulated secretory pathway) Vesicle transport is a multi-step process 2. Formation of coated transport vesicle… 3. Targeting and docking to specific compartment… SNAREs and Rabs Target compartment 1. Formation of coated buds… (ATP, GTP, and cytoplasmic protein factors…) Donor compartment GTP GTPgS GDP + Pi 4. Uncoating… Sar 1 Coat proteins (“COPs”) GTPgS and other non-hydrolyzable GTP analogs block uncoating, resulting in accumulation of docked, coated vesicles GTP hydrolysis by Sar1 is required for uncoating Vesicle transport is a multi-step process 2. Formation of coated transport vesicle… 3. Targeting and docking to specific compartment… SNAREs and Rabs Target compartment 1. Formation of coated buds… (ATP, GTP, and cytoplasmic protein factors…) GTP GDP + Pi 4. Uncoating… Sar1 GEF and Sar1 Donor compartment Coat proteins (“COPs”) GEF in donor membrane promotes nucleotide exchange, activating Sar1 @ ER, (ARF @ Golgi) and promoting coat assembly… GTP hydrolysis serves as “timer” delaying uncoating (GAP in target membrane?)… GTPase “cycle” provides directionality to vesicle coating/uncoating Vesicle transport is a multi-step process 2. Formation of coated transport vesicle… 3. Targeting and docking to specific compartment… SNAREs and Rabs Target compartment 1. Formation of coated buds… (ATP, GTP, and cytoplasmic protein factors…) GNRP/GEF and Coat recruitment GTPase Coat proteins (“COPs” Donor or “coatomer”) GTP GDP + Pi 4. Uncoating… Coat recruitment GTPase compartment 5. Fusion… SNARE plus other fusion proteins SNAREs are necessary for membrane fusion Much still to learn!!! ECB 15-21 SNAREs bring two membranes into close apposition Lipids flow between membranes - fusion Other proteins cooperate with SNAREs to facilitate fusion and to pry SNAREs apart Lecture 16 Vesicle transport and targeting in the secretory pathway COP coated vesicles SNAREs Protein sorting/targeting Secretion - Golgi to plasma membrane Retention in ER Golgi to lysosome How are proteins sorted to appropriate vesicles so that they are transported to proper location? What are the address label? Two secretory pathways; constitutive and regulated Default pathway for ER/Golgi proteins If no address label, then secrete ECB 15-28 Signal required to trigger secretory granule fusion Inside lumen is equivalent Example - neurotransmitter to outside of cell release However, recent data suggests there may be ER exit sequences.. For now, consider secretion default 15.9secretory_pathway.mov Regulated secretion Secretory granules containing insulin in pancreatic cells Signal for release is elevated glucose levels in blood If secretion is default, how are resident ER proteins retained? They aren’t! Ex: BiP is a member of the HSP70 family that functions in the ER… BiP KDEL Constituitive secretion KKXX KDEL-R Secretory granule Regulated secretion ER CGN C, M, T Golgi TGN Plasma membrane Outside BiP escapes from ER and must be “retrieved” from the Golgi… C-terminal KDEL in BiP sequence functions as retrieval signal… KDEL-receptors in Golgi direct retrieval/recycling… KKXX at C-terminus of KDEL-R binds COPI coat and targets back to ER… Summary so far of protein targeting, revisited… Protein targeting Secretion/membrane proteins Cytoplasm Signal sequence (hydrophobic a-helix) Vesicle targeting RER See ECB figure 14-5 Default KDEL (soluble proteins) KKXX (membrane proteins) Golgi Secretory vesicles (regulated secretion) Default ? (constituitive secretion) Plasma membrane Transport Lysosomes Retrieval How are proteins targeted to the lysosome? Lecture 16 Vesicle transport and targeting in the secretory pathway COP coated vesicles SNAREs Protein sorting Secretion - Golgi to plasma membrane Retention in ER Golgi to lysosome How are proteins sorted to vesicles leaving TGN for lysosome? Lysosomes degrade and recycle macromolecules… ECB 15-34 Lysosomes in plant and animal cells contain acid hydrolases (hydrolytic enzymes) for degrading/recycling macromolecules pH of lumen is about 5 - acidic! How are hydrolases and other proteins targeted to lysosomes? I-cell disease helped decipher the signal for targeting proteins to the lysosome • Recessive mutation in single gene… • Fibroblasts of patients contain large inclusions (I-cells)… • Lysosomes lack normal complement of acid hydrolases… • All lysosomal enzymes secreted (secretion is the “default” fate for proteins in the ER-Golgi pathway)… • Lysosomal enzymes of “wild-type” (normal) cells are modified by phosphorylation of mannose on oligosaccharide (forming mannose-6phosphate)… • Lysosomal proteins of I-cells lack M-6-P… • Lysosomal targeting signal resides in carbohydrate! Mannose-6-P targets proteins from Golgi to lysosome Cis Golgi Network (CGN) Addition of M6P RER Lysosomal hydrolase (precursor) Trans Golgi Network (TGN) Transport via clathrin-coated vesicles to… Lysosome Clathrin coat Uncoupling Mature (pH 5) hydrolase M6P receptor Removal of phosphate & proteolytic processing… M6P receptor recycling back to Golgi Addition of M6P to lysosomal enzymes in cis-Golgi M6P receptor in TGN directs transport of enzymes to lysosome via clathrincoated vesicles Patients with I-cell disease lack phosphotransferase needed for addition of M6-P to lysosomal proteins in fibroblasts… secreted… Protein targeting, revisited Protein targeting Secretion/membrane proteins Cytoplasm Signal sequence (hydrophobic a-helix) RER Vesicle targeting Default or signal? KDEL (soluble proteins) KKXX (membrane proteins) Golgi Secretory vesicles (regulated secretion) Default or signal? (constituitive secretion) Plasma membrane See ECB figure 14-5 Transport M6P Lysosomes Retrieval Next lecture: endocytosis and clathrin coats