Membrane Traffic II Flashcards
what are the major takeaways from lecture 1?
- proteins are co-translationally translocated to the ER
- proteins are co- and post-translationally modified
- topology of proteins determined at the ER (lumen of organelles is topological equivalent of the extracellular space)
- protein trafficking requires membrane budding and fusion
- endocytosis leads to recycling and degradation of proteins
what do you need to target cargoes to a particular membrane, like the plasma membrane or the endolysosomal system?
signals on the proteins
advantages of vesicular transport
- proteins can move through different organelles, but only need to cross a membrane once (ER translocation)
- sequential processing events can be compartmentalized
challenges imposed by vesicular transport
- cargoes need to be targeted to specific organelles
- membranes can fuse and divide (energetically unfavorable) –> need a lot of ATP
basic principles of vesicle transport
-compartments with donor membrane and need to concentrate cargoes into these vesicles that bud off compartments –> vesicles targeted to the next compartment and you have to fuse vesicle onto the Golgi membrane
-once you have topology of proteins it’s conserved
what are the steps to vesicular transport?
- budding- requires coat proteins, membrane binding proteins, cargo receptors, and small GTP binding proteins
- targeting- tether proteins, targeting receptors, and small GTP binding proteins
- fusion- “fusion proteins” and disassembly factors
-if you go into the ER compartment to Golgi, soluble proteins and receptors in budding part then gets concentrated for budding that gets assisted by coat proteins and incorporates other proteins like SNAREs and GTPase into the vesicles
-vesicles are budded off from compartment and transport to next compartment and tethering protein is incorporated and tethers protein to the target membrane
-vSNAREs and tSNAREs allow you to fuse vesicles to particular locations
ER export
-soluble and transmembrane proteins- have signals that bind receptors and transmembrane receptors have ER exit signals that bind adaptors that bind coat proteins
-generate vesicles through additional proteins
-once vesicles are made, GTPase activity with GTP hydrolysis occurs and coat proteins disassemble (how membrane budding occurs)
different coats are used at different steps
-COPII- mediates ER budding
-COPI- between Golgi and retrograde trafficking to the ER plus trans-Golgi network
-clathrin- endocytosis
targeting and fusion machinery
-vSNAREs on vesicle and on target compartment you have tSNAREs that make a pair that is quite specific –> need specific pair to line up and mediate fusion (confer specificity for fusion events)
-after fusion with SNARE assemblies, cis-SNARE has to be disassembled –> cis-SNARE complex is one of the tightest you find in the cell so it requires energy and is mediated by the NSF protein
-ATPase unwinds the SNARE complex (chaperone) that unwinds SNARE proteins for next vesicle fusion event
Golgi complex- specialized function in each cisternae
-one of the main functions is to modify the proteins –> as it comes from the ER, proteins are sorted and glycosylation occurs for proteins to be processed
-different sugar modifications that happen
-glycosylated proteins have galactose and NANA added- important for distinction of self
-O-glycosylation- added hydroxyl group to Serine and Tyrosine residues
what are the two places the proteins can go from the Golgi?
- plasma membrane (default pathway)- if there’s nothing happening to proteins after, they go to the PM
-they could be regulated but otherwise you can have exocytosis that happens at the plasma membrane constitutively - endo-lysosomal system
delivery of lysosomal proteins to lysosomes
-lysosomes are important for degradation of proteins and lipids and act as signalling hub
-need to send all these enzymes to lysosomes and membrane trafficking pathway is the way to do it
-if you cannot send proteins to lysosomes, you get diseases liek Gaucher’s disease, Tay-Sachs disease, and Mucolipidosis II
what do you need on lysosomal proteins?
-they must be M6P tagged
-in one of the mannose molecules, GlcNac phosphate is added to the sugar groupa t the mannose-6 position (M6P) then GlcNAc gets removed for only phosphate to stay
-once you have M6P receptor in Golgi, they will be sorted at the trans-Golgi complex and buds whatever is bound to the M6P receptor and sends to lysosomes
lysosomal proteins are recognized by GlcNAc phosphotransferase via the signal patch
-specificity to structure of proteins
-if proteins can bind the GlcNAc phosphotransferase, the first step happens
-Glc-NAc-phosphate is added to the M6P and then you have intermediate protein then phosphodiesterase removes the GlcNAc then you have phosphate group attached to mannose 6 –> sent to lysosomes
M6P-tagged lysosomal proteins bud off from the TGN and delivered to late endosomes
-happens in trans-Golgi network where you have M6P tagged proteins that bind M6 receptor and sorted at the trans-Golgi network with clathrin and adaptors
-send to late endosomes in the lysosomal system and through maturations and infusion, they get delivered to lysosomes
-despite being tagged, proteins can escape from Golgi and go to other pathways
what do you do if you have an issue with the enzymes?
if you have a problem with one of the enzymes, you can use enzyme replacement therapy of M6-tagged enzyme and put it in the extracellular space and it will bind to the M6 receptors and through endocytosis and maturation of endosomes it gets to lysosome
sorting to organelles signals are the key…only lysosomal targeting involves the ER
-all the proteins targeted to mitochondria, peroxisome, and nucleus do not require membrane trafficking pathways
-all the proteins targeted to these structures are made in the cytoplasm but do require some chaperones
signals for ER localization
-retain proteins in the ER and Golgi –> requires certain signals on the protein
-you have a soluble protein that may function in the ER and they have sequence KDEL and allows you to keep proteins in the ER
-if it’s a transmembrane protein, the signal is going to be cytoplasmic side with Dilysine motif on the C-terminus tail
-sec61 protein has this signal to allow the protein to stay in the ER
-ER retention signals- even with signal sequences, they can still be trafficked to the Golgi
yeast secretory pathway
-knew there were compartments in yeast and vesicles sent between them to the Golgi to vacuoles or plasma membrane
-mutagenesis screen- isolated one mutation that caused secretion of soluble protein
the first secretion (Sec) mutant isolated
-at higher temp of 36 degrees, proteins became defective and couldn’t secrete proteins –> saw an accumulation of vesicles
-cells are heavier since they’re constipated
genetic screen scheme for sec mutants
-centrifuge to spin down cells –> they go down to lower sucrose gradient band
-upscale genetic screen and saw which came to heavier locations
-mapped 188 strains onto genes and found 23 genes
EM determined the exact nature of secretion defect
-no vesicles form from the ER and the ER becomes bloated –> see accumulation of vesicles in the cytoplasm that can’t fuse with PM
yeast secretory pathway is likely regulated by gene products
isolated several genetic factors important for membrane trafficking
jim rothman
isolated proteins responsible for fusion at the plasma membrane
N-ethlymaleimide (NEM) blocks vesicle fusion at the Golgi membrane
-used drug that blocks fusion of vesicles to the Golgi (NEM) and you see Golgi with a lot of vesicles associated without fusion
-if you can purify the components that are required for this process using NEM, might be able to tell what proteins may be acting for fusion
-NEM-sensitive factor (NSF)- cytosolic protein and purified from Golgi membrane with washing off NEM and adding in ATP to dissolve NSF then purified it
-added NSF onto purified Golgi and vesicles then did in vitro assay
NSF required for fusion of cargo vesicles to target compartments
-you can fuse vesicles to Golgi in vitro
-NSF is encoded by sec18, suggesting gene products do mediate fusion events
performed Co-IP experiment with brain extract
-came in with antibody against NSF and pulled everything that binds to it –> called it soluble NSF-attachment proteins (SNAP) and SNAP receptors
-trypsinized it and sequenced to find proteins encoded by 3 different genes: syntaxin, SNAP-25, and synaptobrevin
purification of neuronal SNAREs from E. coli (liposome assay)
-recombinant proteins in bacteria –> purify them –> put into liposomes and see if they actually fuse
-knew three components: syntaxin, SNAP-25, and VAMP
-purified from E. coli and put in liposomes
-see v-SNARE as donor compartment with acceptor vesicle –> have to have v-SNAREs on one side and t-SNAREs on the other
-provided evidence that SNAREs mediated fusion
tetanus toxin to ask whether synaptic vesicle secretion is inhibited (SNARE specific proteases)
-when applied to neurons, showed that you cannot secrete neurotransmitter and showed that SNAREs mediate vesicle-membrane fusion
bacterial toxins target the vesicle docking and fusion machinery of neurons
-botox- blocks excitation and don’t have tight muscles in the face- affects synaptic transmission by cleaving SNARE proteins
SNAREs are required for docking and fusion
-SNAREs mediate docking and infusion of vesicles to target membrane
-tethering proteins that bind GTPase and how you can target the vesicles to certain locations
-vesicles have different Rab-GTPases associated with them and there’s a Rab affector or tethering proteins on target membrane that binds them
-SNARE proteins get associated once proteins are bound to target membrane and mediates fusion events –> exocytosis
-NSF unwinds cis-SNARE complex, which requires ATP to unwind
specialized SNAREs mediate fusion at each trafficking step in the cell
confers specificity to the target membrane
what does budding require?
- coats
- adaptors
- membrane bending proteins
coat proteins bud vesicles off from the membranes
-COPII in ER, COPI in the Golgi, and Clathrin for the trans-SNARE complex and plasma membrane
-concentrated cargoes by adaptor proteins –> adaptor proteins bind cargoes and what codes to particular membrane
-promotes membrane budding events and gets vesicles released
GTP hydryolysis leads to uncoating
-in the case of COPII and COPI coats, GTP hydrolysis with GTPase the coats get disassembled
-ATPase gets recruited to Clathrin and removes it
what is endocytosis important for?
nutrition, defense, signaling, and homeostasis
what are the different types of endocytosis?
phagocytosis, macro-pinocytosis, clathrin-mediated endocytosis
clathrin-mediated endocytosis
-have adaptor proteins that bind the cargoes –> adaptors then recruit clathrin to the site –> promotes budding of the membrane to make vesicles that get cut from the plasma membrane
-ATPase then comes in and disassembles clathrin
-coat proteins (clathrin) do not bind cargoes and membranes directly
EM with yolk uptake in oocytes in the ovary of the mosquito with apparent electron-dense coat
a lot of yolk uptake and looked to see coats that budded on plasma membrane
Barbara Pearse purified clathrin-coated vesicles from the brain and looked at it with negatively stained vesicles
see coat on vesicles and named it clathrin
familial hypercholesterolemia: high cholesterol level in the blood
-interested to know what was causing the buildup of cholesterol?
-sequenced the LDL receptor and found mutation in the cytoplasmic tail
-in Tyrosine residue it was mutated to be Cysteine and caused issues
-isolated fibroblasts from patients and looked at endocytosis of receptors
-in healthy fibroblasts, endocytosis occurring normally with LDL that gets sorted into coated pits
-in patients, the clathrin-coated vesicles never got concentrated into pits and were all over the plasma membrane –> mutation in the cytoplasmic tail is important
adaptor protein complex binds clathrin and cargoes
-adaptor protein complexes bind Tyrosine motifs and if you have one of these on the cytoplasmic tail, they can get sorted into clathrin-coated pits
-AP2 works on the plasma membrane
-binding of AP1 and AP3 determines where cargoes get sent –> if it doesn’t bind strongly to AP1, it gets sorted to AP3 and goes to certain locations
-affinity decides what compartments proteins go to
the endocytic pathway leads to degradation or recycling
-compartments are acidified- pH in extracellular space is 7.4 but in endosomes its pH is 6 and in lysosomes pH is 5 —> ligands and receptors get dissociated and can get recycled
Ex. LDL receptor with LDL ligand with cholesterol –> LDL receptor binds it and sorts it into clathrin-coated vesicles and goes to endosomes
-in the early endosomes, disassociation of the ligand LDL from LDL receptor and LDL receptor can be recycled back to plasma membrane whereas ligand goes all the way to the lysosome and proteins get degraded but cholesterol stays
-NPCI and 2 take up free cholesterol and put it into the lysosomal membrane and re-distributes into the ER and plasma membrane
-disassociation of ligands and receptors require different pHs
endosomes can be matured into late endosomes to make multi-vesicular bodies
buds membrane into the lumen or the organelle or endosomes –> making interlumenal vesicles
ESCRT proteins bud vesicles into the lumen of endosomes
-secreted out as exosomes or degraded by lysosomes
-complex pushed membrane into the lumen of organelle
