L13 protein transport (Tfk2)

• we need a Rab GTPase to provide specificity for where the vesicle needs to go and attach to acceptor membrane, as well as a SNARE to tell vesicle where to fuse on the membrane for vesicle trafficking
• golgi is organized into a stack of membranes: cis, medial, trans
• two models explain how golgi vesicles go from one side to another side of the golgi:

  old model -> assumes vesicles just pass through and golgi stays the same

  new model -> an intermediate compartment A is formed where vesicles fuse that becomes the cis golgi. the trans golgi disappears into new vesicles, and new intermediate compartment B arrives becoming the new cis golgi and intermediate compartment A becomes the medial golgi. each layer matures to become the next layer
• proteins produced in the ER have the PTM of adding n-linked glycans. they are modified to remove the glucose, and in the golgi, the manoses can be removed as well, and we can add other proteins to these oligosaccharides.
• n-linked glycosylation occurs in the ER while o-linked glycosylation occurs in the golgi. motif for n-linked glycosylation is Asn-X-Ser/Thr; motif for o-linked glycosylation is hydroxyl groups of serine and threonine
• glycosylation in the golgi promotes protein folding by acting as a sequential modification that allow for a code that progresses folding of the protein. folding intermediates are additionally more soluble (preventing aggregation). saccharides (added through glycosylation) protect from proteases and act as protective coat that allow for signaling pathways.
• some plasma membrane proteins are synthesized as large protein precursors and have an inhibitory sequence that needs to be cleaved. proprotein convertases recognize specific amino acid sequences and cleave it, allowing the PM protein to be active. examples of these PM proteins include proinsulin (loop section bound by disulfide bonds of proinsulin cleaved) and ATF6 (needs to be cleaved in golgi to upregulated target genes for UPR).
• Rab proteins provide specificity for proten transport. when bound to GTP, Rab is active. when GDP is bound, it is inactive. there are many different types of Rab proteins

  • some assist in cargo selection and coat formation
  • others connect vesicle to motors on cytoskeleton for transport
  • some will tether vesicles to acceptor membranes
  • others recruit SNARE fusion proteins

  the effectors of the Rab proteins have the function, not the Rab protein themselves
• Ras (NOT RAB) anchored into membranes via amphipathic helices facing lumen or cytosol. Rab has prenyl lipid groups at c-terminus that become prenylated via cysteine motifs, these are Rab membrane anchor proteins.

  • in GDP bound state, no prenyl groups will attach. GDI and GDF hide these hydrophobic prenyl groups
  • in GTP bound state, prenyl groups attach to Rab and membrane (anchoring Rab).
  • rab-effector proteins become attached to the membrane via Rab-GTP.
• specific GEFs (GDP -> GTp) will work as an anchor on the membrane for the Rab-GTP. effector proteins have the function, not Rab-GTP. functions of effector proteins include

  • attach vesicle to motor proteins
  • tether vesicle to target membrane
  • activate phosphatidyl inositol kinases
• vesicle rab cycle is:

  1. Rab-GDP attached to GDF and GDI (prenyl groups hidden).
  2. GEF attached to membrane making Rab-GTP. prenyl groups insert Rab into membrane
  3. when vesicle pinched off, Rab may interact with effector that binds effector to transport molecules, etc.
  4. Rab tethers the vesicle to the compartment, recruits a SNARE for fusion of vesicle
  5. once tethered, hydrolysis of GTP makes a soluble Rab-GDP to be recycled and reused
• cytoskeleton creates the ability to move cargo from one side to another. it is made of protein filaments (actin) and microtubules (tubulin).

  • actin attach many molecules, clustered at PM, in neurons around synapses. microtubules attached near centrosome, in neurons around axons and dendrites. actin filaments and microtubules are connected to each other and they anchor the organelles and the plasma membrane.
• motor proteins move vesicles around cell via cytoskeleton. myosin is the motor protein for actin; dyneins (AAA-ATPase) and kinesins are motor proteins for microtubules. they all depend on ATP, have no targeting specificity.
• many Rab effectors (other proteins that work with Rab) are tethers. tethers are the first determinants of vesicle targeting specificity.
• types of tether effectors of Rab proteins:

  • coiled-coil (two alpha helices) tethers in golgi, they are always there, don't get recycled. have multiple Rab binding sites
  • multisubunit tethers that localize in different regions of the pathway (ER -> golgi; golgi -> PM, endosome and lysosome)
• types of multisubunit tether effector proteins of Rab proteins:

  1. ER to golgi have TRAPPI (GEF, effector is coiled-coiled tether p115)and TRAPPII. have 3 domains, one for vesicle, one for GEF and one for SNARE
  2. endosome has Rab5 (tether is CORVET), lysosome has Rab7 (tether is HOPS)
  3. ER and PM have catcher family with exocyst (8-protein complex with GARP, COG, Dsl1) at plasma membrane
• there is a clustering of the tethers, that form the landing site for vesicle.
• Rab5 effectors in endosome have GEF (produces more Rab5-GTP in local area on membrane) or PI kinase activity (additional binding sites) during early endosome creating a clustering of tethers.
• endocytosis is the process of engulfing material from the extracellular environment.

  • CCV take cargo from exterior bringing it inside (no specificity of cargo).
  • early endosome is made by diffusion of different vesicles, has Rab5.
  • early endosome matures into multivesicular body (MVB) resulting in endoslysosome and in lysosome components are degraded into monomers. late endosome has Rab 7
• Rab5 becomes Rab7 through the early and late endosome process.
• in the endosome, there are GEFs specific for Rab5. Rab5 has an effector that is a GEF for Rab7. effector of Rab7 is GAP for Rab5.

  • when Rab5 is active (prenyl groups shown), it attaches to membrane bringing its GEF-Rab7 effector.
  • Rab7 then becomes activated, bringing the Rab5 GAP effector.
  • Rab5 is then inactivated (no prenyl groups shown), disassociating it from the membrane.
• Vesicle trafficking requires two types of molecules:

  • Rab GTPase provides specificity for vesicle destination and attachment to acceptor membrane
  • SNARE guides vesicles to fuse on the membrane
• Proteins in vesicle traffic use Rab GTPase for specificity, SNARE for fusion guidance
• Old model of Golgi function: assumes all Golgi membranes are the same (COPI and COPII), while new model shows maturation of each layer from ER to vesicles to intermediate compartments, with movement from cis to trans
• In the Golgi, proteins undergo modifications like removing glucose and manoses, and adding other proteins to oligosaccharides
• n-linked glycosylation occurs in the Golgi on serine and threonine residues, creating diverse oligosaccharide combinations
• Post-translational modifications (PTMs) in Golgi promote protein folding, solubility, prevent aggregation, and act as signaling pathways
• Proprotein convertases in the Golgi cleave inhibitory parts of proteins, activating them for their intended function
• Rab proteins, when bound to GTP, are active and provide specificity in vesicle targeting, recruiting SNARE fusion proteins for membrane fusion
• Rab proteins have prenyl lipid groups attached at their C-terminus, allowing membrane anchoring in the GTP-bound state
• Rab-GTP works through effector proteins, which may attach vesicles to motor proteins, tether vesicles to target membranes, and activate other GEFs for more Rab-GTPs
• The vesicle Rab cycle involves Rab attached to GDF and GDI in the GDP state, then GEF on the membrane makes Rab-GTP, allowing insertion into the membrane
• In the vesicle Rab cycle:

  • Rab is attached to GDF and GDI in the GDP state, which is a soluble state
  • GEF on the membrane creates Rab-GTP, helping insert Rab into the membrane
  • Rab interacts with an effector after vesicle pinching off, aiding in transport to the delivery compartment
  • Rab tethers the vesicle to the compartment, recruiting SNARE for vesicle fusion
  • After vesicle fusion, Rab is hydrolyzed to RabGDP, becoming soluble for recycling and reuse
• The cytoskeleton, made of actin filaments and microtubules, allows cargo movement within cells:

  • Actin filaments are found in fibroblasts and synapses, while microtubules are near the centrosome
  • Actin filaments and microtubules anchor organelles and the plasma membrane
• Motor proteins like myosin, dyneins, and kinesins move vesicles on the cytoskeleton, depending on ATP for energy
• In vesicle tethering:

  • Rab effectors are tethers, binding to a specific vesicle Rab and a membrane site
  • Tethers are crucial for vesicle targeting specificity
• Types of tethers include:

  • Coiled-coil tethers in the golgi and endosome, maintaining organization and connecting vesicles
  • Multisubunit tethers in the secretory pathway, like TRAPPI and TRAPPII for ER to Golgi transport
• In the endocytosis pathway:

  • Clathrin coat vesicles transport cargo into the cell, with early endosomes maturing into late endosomes and lysosomes for degradation
  • Rab5 is in early endosomes, while Rab7 is in late endosomes
• In Rab cascades:

  • A specific guanine exchange factor for Rab5 is present in the endosome
  • Rab5 is replaced by Rab7 through effectors, with a GAP for Rab5 hydrolyzing its GTP to GDP
• NSF binds to the SNARE complex through an adaptor protein (alpha SNAP) to unwind the SNARE helices