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 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 (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 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. 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 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

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 However, recent data suggests there may be ER exit sequences.. For now, consider secretion default 15_28_trans_Golgi_net.jpg ECB 15-28 Signal required to trigger secretory granule fusion Example - neurotransmitter release Inside lumen is equivalent to outside of cell 15.9-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? 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 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…

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 See ECB figure 14-5 Transport Retrieval Next lecture: endocytosis and clathrin coats