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, NR2A-D, 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 A shows the accumulation of coated vesicle intermediates when transport is blocked with non-hydrolyzable GTP analogues. B shows a COPI coated vesicle.

Cop coated vesicles contain many proteins COP proteins More COP proteins “cargo” Lipid bilayer Sar1 COPII-coated vesicles - ER to Golgi- SarI 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 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 In cytoplasm, large amount in “off” form Sar1 GTPase GTP Sar1 GTPase GDP GDP GTP Bound to membrane “on” “off” GEF 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 Bound Unbound B A + GTP GDP + Pi A paradox: High-affinity/high-specificity = stable… Energy input is required to dissociate high-affinity complexes… (Example: to remove Sar 1 from membrane) Polymer dynamics: Actin (ATP), Tubulin (GTP) Dynamin (GTP) Motors: Myosin (ATP), Dynein (ATP) Kinesin (ATP) Signaling: Heterotrimeric G proteins (GTP) Ras family (GTP) 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)

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…) Coat proteins (“COPs”) Donor compartment Inhibitors such as GTP-g-S and NEM have been used to map out the steps involved in vesicle transport: Formation of coated buds (requires ATP and cytosol); Formation of coated vesicles (requires ATP and cytosol); Transport and docking to the target membrane; Uncoating (requires GTP hydrolysis; blocked by GTP-g-S); and Fusion (blocked by NEM).

The Snare hypothesis: v- and t-SNAREs target transport vesicles to the correct membrane Budding Uncoating, targeting and docking Cargo t-SNAREs v-SNAREs Specific pairing of receptors known as V-snares (in the vesicle membrane) and T-snares (in the target membrane) ensure the correct targeting of membrane vesicles. Other proteins, such as SNAPs, NSF, and the small GTPase Rab play important roles in membrane fusion. 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 Botulism B Botulism C Tetanus SNAP25 (t-SNARE) VAMP (v-SNARE) Syntaxin (t-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 GTP GDP + Pi (ATP, GTP, and cytoplasmic protein factors…) 4. Uncoating… GTPgS 1. Formation of coated buds… Sar 1 Donor compartment Coat proteins (“COPs”) Inhibitors such as GTP-g-S and NEM have been used to map out the steps involved in vesicle transport: Formation of coated buds (requires ATP and cytosol); Formation of coated vesicles (requires ATP and cytosol); Transport and docking to the target membrane; Uncoating (requires GTP hydrolysis; blocked by GTP-g-S); and Fusion (blocked by NEM). 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 GTP GDP + Pi (ATP, GTP, and cytoplasmic protein factors…) 4. Uncoating… 1. Formation of coated buds… Sar1 GEF and Sar1 Donor compartment Coat proteins (“COPs”) Inhibitors such as GTP-g-S and NEM have been used to map out the steps involved in vesicle transport: Formation of coated buds (requires ATP and cytosol); Formation of coated vesicles (requires ATP and cytosol); Transport and docking to the target membrane; Uncoating (requires GTP hydrolysis; blocked by GTP-g-S); and Fusion (blocked by NEM). 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 GTP GDP + Pi (ATP, GTP, and cytoplasmic protein factors…) 4. Uncoating… 1. Formation of coated buds… Coat recruitment GTPase GNRP/GEF and Coat recruitment GTPase Donor compartment Coat proteins (“COPs” or “coatomer”) Inhibitors such as GTP-g-S and NEM have been used to map out the steps involved in vesicle transport: Formation of coated buds (requires ATP and cytosol); Formation of coated vesicles (requires ATP and cytosol); Transport and docking to the target membrane; Uncoating (requires GTP hydrolysis; blocked by GTP-g-S); and Fusion (blocked by NEM). 5. Fusion… SNARE plus other fusion proteins

SNAREs are necessary for membrane fusion Much still to learn!!! ECB 15-21 15_21_membr_fusion.jpg 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 However, recent data suggests there may be ER exit sequences.. For now, consider secretion default 15_28_trans_Golgi_net.jpg Signal required to trigger secretory granule fusion Example - neurotransmitter release Inside lumen is equivalent to outside of cell 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 KKXX KDEL-R Constituitive secretion Secretory granule Regulated secretion Plasma membrane ER CGN C, M, T Golgi TGN 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… Secretion/membrane proteins Protein targeting Cytoplasm RER Signal sequence (hydrophobic a-helix) Vesicle targeting Golgi Default KDEL (soluble proteins) KKXX (membrane proteins) Lysosomes ? Secretory vesicles Plasma membrane Default A schematic summary of protein targeting and vesicle trafficking in a typical eukaryotic cell. (regulated secretion) (constituitive secretion) See ECB figure 14-5 Transport Retrieval How are proteins targeted to the lysosome?

How are proteins sorted to vesicles leaving TGN for 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… The lysosome of animal cells contains hydrolytic enzymes for degrading/recycling macomolecules. The vacuole of plant cells performs many of the functions of the animal lysosome, as well as regulating turgor pressure. 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-6-phosphate)… 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) Trans Golgi Network (TGN) Addition of M6P Transport via clathrin-coated vesicles to… Lysosome Clathrin coat Uncoupling (pH 5) Mature hydrolase RER Lysosomal hydrolase (precursor) M6P receptor Removal of phosphate & proteolytic processing… M6P receptor recycling back to Golgi Lysosomal proteins are modified by the addition of M-6-P to their polysaccharides in the cis-Golgi. The trans-Golgi and/or TGN contains M-6-P receptors, whioch bind lysosomal proteins. These receptors are recruited into clathrin-coated pits/vesicles on the TGN, and transported to the endosome, and thence to the lysosome. Addition of M6P to lysosomal enzymes in cis-Golgi M6P receptor in TGN directs transport of enzymes to lysosome via clathrin-coated vesicles Patients with I-cell disease lack phosphotransferase needed for addition of M-6-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) EE Trans face Golgi RER LE COPII Lysosome COPI EE AP1 (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 Secretion/membrane proteins Protein targeting Cytoplasm RER Golgi Plasma membrane Signal sequence (hydrophobic a-helix) Vesicle targeting Default or signal? KDEL (soluble proteins) KKXX (membrane proteins) Secretory vesicles M6P Default or signal? A schematic summary of protein targeting and vesicle trafficking in a typical eukaryotic cell. (regulated secretion) (constituitive secretion) Lysosomes Transport Retrieval

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-dep- dent 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.