Book Notes: Membranes Part 3

Side note: I generally don't like to follow the random given page numbers on the syllabus but reorganize it so that it goes in page order.  In this topic, however, the syllabus happens to go in page order.  So hooray!

• transport vesicles: membrane bound containers that move lumenal residents between membrane bound areas (avoids transmembrane traffic)
• cargo: soluble proteins that the vesicles carry
• export of secretory stuff and import of endocytosed stuff balance out the membrane transport going out and in.
• 3 ways of studying transport mechanisms:
• cell-free organelle only systems: isolate Golgi stacks, observe transport vesicles budding and transporting between pancake stacks
• put normal Golgi stack and mutant Golgi stack together (mutant lacks N-acetylglucosamine transferase I, so, it can't glycosylate proteins)
• mutant has a viral protein that needs a sugar, normal Golgi has the enzyme but no protein to modify
• label the sugar.  if the sugar gets added to the protein, the protein must have been transported from mutant Golgi to normal Golgi, which occurs only with ATP and cytosol
• fractionation isolates proteins involved in transport
• genetics: identify some temperature conditional mutant proteins necessary for transport (function well at low temperature, deforms at high temperature)
• however, if enough substrate is present, it will overcome mutant's deformity and catalysis still occurs as normal
• what is the substrate?  we put in tons of random DNA fragments in mutant cells and turn up the heat
• any cells with lots of the correct and intact gene on random fragments will overproduce whatever protein that gene encodes
• having lots of protein will override the high temp protein deformity and only those cells will survive the oven
• isolate DNA from the surviving cells to decode what the protein substrate was
• GFP fusion: attach GFP to POI, transfect cell with the corresponding cDNA, and watch GFP move between organelles
• use FRAP or FLIP to determine rate of diffusion/transport
• coated vesicles: membrane that is about to pinch off receives a special protein coat on the cytosolic side
• the coat proteins selectively gather the membrane molecules that will be transported and molds the vesicle shape by assembling in curved lattice basket
• clathrin-coated: transport between Golgi, plasma membrane, endosomes
• clathrin: 3 large and 3 small chains that make a curvy 3 way boomerang shape "triskelion", spontaneously assemble into soccer ball shape with pentagons and hexagons
• adaptor proteins form a second coat btwn clathrin and membrane, bind clathrin coat and specific transmembrane proteins whose lumenal side binds the cargo (cargo receptors)
• different adaptor proteins choose different cargo-receptors and affect how the clathrin cage is formed, so each vesicle with different cargo also looks slightly different on the outside
• COPI-coated: bud from Golgi to Golgi or ER
• COPII-coated: bud from ER to Golgi
• both COP coats function similarly to clathrin coats
• retromer: coat that form as patches on endosomes to return acid hydrolase receptors (e.g. mannose-6-Pi receptor) to Golgi
• binds only when 3 conditions are met simultaneously (coincidence detector)
• it can bind to cargo receptor cytosolic tails
• it can bind to curved phospholipid bilayer
• it can bind to phosphorylated phosphoinositide on the surface
• binds as a dimer, stabilizes curvature so more can bind
• leads to budding of a vesicle that returns to the Golgi
• phosphoinositide (PIP): phosphorylated inositol phospholipids (at 3', 4', or 5' position of sugar head group)
• phosphatidylinositol (PI) is the dephosphorylated version
• how it is phosphorylated is distinct to each organelle (each organelle has different set of PI and PIP kinases and PIP phophatases)
• PIPs are markers identifying different organelles
• proteins involved in transport bind only to correct PIP
• local regulation of PIP production thus controls amount of incoming transport
• dynamin: cytosolic protein, assembles as ring around pinch off part as clathrin vesicle buds
• binds to PIP phosphorylated at 4' and 5' and has a GTPase domain
• recruits other proteins to bend the membrane to fuse off and/or to change the lipid composition to help the pinching process
• a pinched off vesicle loses the clathrin coat and a PIP phosphatase dephosphorylates all PIPS (so adaptor proteins fall off)
• Hsp70 chaperone spends ATP to peel off the clathrin
• regulated by auxillin (who activates the ATPase domain) to prevent clathrin removal before budding is complete
• Coat-recruitment GTPase: monomeric GTPases, include Arf (who makes COPI and clathrin coats at the Golgi) and Sar1 (who makes COPII at ER)
• exist in high concentrations of GDP-inactive form in cytosol
• the appropriate GEF is embedded in the membrane, when the membrane wants to bud, GEF causes the recruitment GTPase to swap for a GTP
• GTP exposes amphipathic helix on the GTPase, so it gets inserted into the membrane bilayer and recruits coat proteins to start budding
• GTP hydrolysis is like timer: Arf and Sar1 will eventually hydrolyze GTP, if bud has not completed by the time GTP is hydrolyzed, then the coat will fall apart and no vesicle will form
• vesicle budding/fusion tends to occur at already curved places (less work for clathrin)
• e.g. plasma membrane is extremely stiff due to cholesterol and cortex, so clathrin has to work extra hard to bend it
• some surfaces don't form spherical vesicles: trans Golgi donates pinched off tubules as transport
• tubules carry more membrane proteins than lumen proteins (high SA to V ratio)
• Rab: protein family of monomeric GTPases that guides vesicle to correct destination
• each organelle has a different Rab
• Rab-GDP is bound to GDI (Rab-GDP dissociation inhibitor) that keeps Rab in the cytosol
• Rab-GTP is actively associated with its organelle's membrane
• membrane bound Rab-GEFS activate specific Rabs on both target membrane and vesicle membrane
• these membrane Rabs bind to Rab effectors, who manage fusion and tethering and all the other modes of transporting the vesicle
• tether proteins and motor proteins are examples of Rab effectors
• process involves a lot of positive feedback
• Rab5-GEF on target membrane activates and recruits Rab5, who anchors to the membrane
• active Rab5 grabs more Rab5-GEF, who recruits more Rab5
• active Rab5 also activates PI 3 kinase, who turns local PI into PIP phosphorylated at 3'
• PI(3)P binds Rab effectors like tethering proteins to catch appropriate incoming vesicle
• SNARE: membrane fusion proteins, exist in organelle specific sets with v-SNARE on vesicle and t-SNARE on target membranes
• single chain of v-SNARE wraps around 2+ chains of t-SNARE to form helix bundle called trans-SNARE complex
• squeeze membranes together, a process accelerated by Rab effectors
• t-SNAREs must be released from inhibitory proteins by Rab and effectors to activate their function
• transport vesicles tend to form only if both appropriate SNARE and Rab proteins are incorporated into the budding membrane
• NSF: protein that moves between membrane and cytosol, spends ATP to unwind the two SNAREs
• virus fusion strategy may be similar to SNARE mechanisms
• membrane bound viruses meet receptors on target cells
• binding exposes a hydrophobic region "fusion peptide" that automatically inserts into membrane bilayer
• these fusion proteins then twist to squeeze and fuse membranes
• Golgi apparatus: site of carbohydrate synthesis, ER products sorting and shipping
• ER exit sites: smooth ER sections where COPII-coated vesicles form
• proteins that need to exit have signals on their sequence to attach to cargo receptors, while cargo receptors have signals on their cytosolic face that attach to COPII
• high concentrations of secretory proteins end up in vesicles without receptors and some ER resident proteins will leak out too
• misfolded proteins are held back in ER by chaperone protein binding, who either cover the exit signal or anchor it down
• misfolded proteins are eventually kicked out into the cytosol to be degraded
• around 90% tend to be thrown out, so cells must make a lot of polypeptides so that some will actually fold properly and function
• serves as early warning system: fragments of whatever gets digested in proteasomes come into the ER, get bound to class I MHC proteins on the lumen, and end up on the plasma membrane outer surface
• T lymphocytes check out the exposed fragments: if they recognize it, it means it's a foreign agent and this cell that's exposing it has been infected by evil stuff ("it's time to die")
• vesicular tubular clusters: fusion of multiple vesicles budding from ER
• homotypic is fusion of vesicles from same organelle (as opposed to heterotypic), requires set of matching SNAREs
• continues as transport to Golgi but buds off COPI-coated vesicles that send fugitives and cargo receptors that helped in budding back to the ER ("retrieval/retrograde transport")
• resident membrane proteins have ER retrieval signals that bind to COPI coats
• most popular is KKXX signal (Lys Lys any any)
• resident lumen proteins have different signals, most popular being KDEL (Lys, Asp, Glu, Leu)
• must bind this sequence to the transmembrane KDEL receptor, who has to cycle back and forth and bind low-concentration proteins in the vesicular tubular cluster but release in the high-concentration interior of ER lumen
• some interface proteins (cargo receptors, SNAREs) don't have signals and just randomly go between ER and Golgi
• kin recognition: ER resident proteins also aggregate in huge clusters too big to enter transport vesicles, general mechanism to organize and retain residents
• Golgi is composed of flattened stacks called cisternae
• cis face is towards ER while trans face is towards plasma membrane
• each stack contains different enzymes for producing different modifications and each enzyme will not accept a substrate unless it has undergone the previous step's modification
• cis Golgi network (CGN): sorting, phosphorylation of oligosaccharides
• cis cisterna: removal of mannose
• medial cisterna: removal of mannose, addition of N-acetylglucosamine
• trans cisterna: addition of galactose, addition of N-acetylneuraminic acid (a sialic acid)
• trans Golgi network (TGN): sulfation of Tyr and carbs, sorting
• all the Golgi resident proteins are membrane bound
• 2 classes of N-linked sugars on glycoproteins
• complex oligosaccharides: original N-linked oligosaccharide is trimmed and gets more sugars
• can have more than 2 of the original N-linked sugars, plus galactose, sialic acid, and fucose
• sialic acid is the only negatively charged sugar in glycoproteins
• high-mannose oligosaccharides: original N-linked oligosaccharide is trimmed but gets no more sugars
• 2 of the original N-acetylglucosamines and lots of mannose
• whether a glycoprotein gets complex or high-mannose depends on if the oligosaccharide is in an accessible position (hidden away means it's probably a high-mannose)
• O-linked glycosylation: same N-acetylglucosamine but attached to hydroxyl of Ser or Thr or hydroxy-Lys
• gets conferred on mucins and proteoglycans
• O-linked proteins get heavily sulfated and become very negative
• both mucins and proteoglycans are secreted (ECM and slimy mucus coating)
• glycosylation helps in folding, sorting, protection, and regulation
• solubilizes proteins in midst of folding to prevent aggregation
• sequential modifications mark progress of folding and mediates binding to chaperones
• lectin modifications guide sorting
• sugar protrusion prevents digestion by proteolytic enzymes
• mucus coats protect against invasion or sugars change antigenic recognition
• sugars also change specificity of regulatory receptors and targets
• transport between stacks occur by 2 ways
• vesicular transport model: Golgi stacks are fixed, while stacks form COPI vesicles to go backward or forward between stacks, either with direction by adaptor proteins or randomly
• cisternal maturation model: arriving vesicular tubular clusters fuse to form CGN, this set of stacks mature to become the cis cisterna, then medial cisterna, and so on
• everything moves continually forward, while vesicles continually backwards to shift the enzymes and return bad stuff
• in reality, it may be combination of both models
• Golgi matrix proteins organize the stack
• cytoplasmic scaffolds give structure and localize Golgi, phosphorylation during mitosis leads to disassembly and fragmentation
• observation that mutation or removal of Golgi membrane proteins still allows Golgi to assemble at right place in right shape, so it must be all on the matrix scaffolds

pg. 749 - 779 (from Chapter 13)

GEF = guanine nucleotide exchange factor

SA = surface area

V = volume

POI = protein of interest

Pi = phosphate