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Protein Trafficking 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 Protein Trafficking - Regulated transport to the trans-Golgi network • Multimeric proteins (e.g., ion channels). - KATP channels = 4 Kir6.1/6.2 subunits with 4 SUR1/2A/2B subunits in ER. - NMDAR = combination of NR1, NR2AD, or NR3A-B subunits. - GABAAR: 16 different mammalian isoforms (α1-6, β1-3, γ1-3, δ, ε, π, and θ), making the total number of receptor combinations = 165; but only ~20-30 functionally distinct receptor types exist. • During GABAAR assembly, chaperones, IgG-bp (BiP) and calnexin, interact with subunits. • Association with ER depends on ER retention signals (KDEL). • Hydrophobic residues. • Exact mechanism of ER retention involves interaction with ER matrix, failure to be recruited for transport, or retrieved from the cis Golgi. • Coatomer proteins (COPS) are involved in the selection of cargo for anterograde (COPII) or retrograde (COPI) transport between organelles. Morphology • Diffusion barrier • Cytoskeleton = actin-spectrin-ankyrin anchors membrane proteins (e.g., Nav channels). • High [protein] crowding. • Soma, dendrites, axon - not 1 continuous structure. • Inhibitory synapses vs excitatory synapses. Development of Polarity • Synapse Formation: - GFP-PSD-95 visualized extension and maintenance of filopodia. - Appeared to be translocated to filopodia as pre-assembled clusters, rather than as accumulating gradually. - But occurs only when the postsynaptic scaffold/signaling complex is already there. - Within 45 min, AMPA and NMDA receptors can be found postsynaptically. Development of Polarity • Axonal Development - Nav channels cluster at the Nodes of Ranvier. - Mechanism of how this occurs is unknown. - In demyelinated axons, some form of anchoring occurs via Ankyrin G within the axon. -In the PNS, paranodal Kv channels appear to cluster initially within nodes prior to lateral diffusion to their final destination. Development of Polarity Dendrite + - + Axon - + + mGluR2 mGluR7 Polarity Signals • Dendrites hydrophobic motifs • Axons – GAP43 Postysynaptic Targeting • mRNA Targeting • Protein targeting via lipid rafts • Specific transport pathways and proteins - GABAA receptors - NMDA receptors - AMPA receptors Transport between organelles is mediated by coated vesicles Clathrin coated vesicles mainly involved in endocytosis 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 • Arf/Sar family of “Coat recruitment GTPases:” COP assembly and vesicle budding… • Rab family: vesicle targeting and fusion (see below) 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 + B Unbound A paradox: High-affinity/high-specificity = stable… Energy input is required to dissociate high-affinity complexes… (Example: to remove Sar 1 from membrane) Translation: Polymer dynamics: IFs (GTP), EF-1/EF-Tu (GTP) Actin (ATP), Tubulin (GTP) EF-2/EF-G (GTP) Dynamin (GTP) Chaperones: Motors: HSP70 family (ATP) Myosin (ATP), Dynein (ATP) HSP60 (ATP) Kinesin (ATP) SRP family: Signaling: SRP54 (GTP), SRP-Ra (GTP) Heterotrimeric G proteins (GTP) SRP-Rb (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 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 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 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 secretory_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? 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… 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… Postsynaptic Removal of Receptors • Specific endocytotic signals leads to recruitment of AP2 in the internalization of the plasma membrane. • APs recruit clathrin, which instigates membrane invagination and endocytosis. • Examples: - tyr-based signals recruit μ subunits of AP2. - dileu-based signals recruit β subunits of AP2. - Arrestin binding to GPCRs facilitate receptor internalization by its ability to assocociate with clathrin and AP2. - Ubiquitin may recruit AP2 or clathrin, release the receptor from anchoring in the membrane, or recruit receptors to the sites for endocytosis. AP2 (rapid) Trans face Golgi EE LE Lysosome RER AP1 EE (rapid) Receptor Endocytosis • Agonist-dependent down-regulation of receptors has been observed for a wide variety of ligands: e.g., GABAA receptors treated with GABA, BDZs, barbs, and neurosteroids; antidepressants and β-adrenergic receptors. • Cell surface receptor number is a balance between 2 competing processes: delivery and removal of receptors. • Synaptic strength is in part, determined by the number of surface AMPA receptors (LTP vs. LTD). BUT… Evidence has shown that in response to NSF-GluR2 interaction, synaptic AMPA receptors are only internalized on the cytoplasmic face of the membrane and are not transported to the soma and degraded in the lysosomes. • Insulin can also cause AMPA receptor down-regulation. Protein targeting, revisited Protein targeting Secretion/membrane proteins Cytoplasm Signal sequence (hydrophobic a-helix) Vesicle targeting RER Default or signal? KDEL (soluble proteins) KKXX (membrane proteins) Golgi Secretory vesicles (regulated secretion) Default or signal? (constituitive secretion) Plasma membrane Transport Retrieval M6P Lysosomes The modulation of synaptic strength by alterations in postsynaptic AMPA receptors. Early in development, most of the glu synapses are ‘silent’ at Vm. This results from the presence of NMDA, but not AMPA, receptors in the postsynaptic membrane. Synapses become activated by a NMDA-depdent process, leading to the recruitment of AMPA receptors. Synaptic may be incr further, in response to high-freq activity (LTP), by the further recruitment of AMPA receptors.