Membrane Traffic II

Question 1

what are the major takeaways from lecture 1?

Answer 1

1. proteins are co-translationally translocated to the ER
2. proteins are co- and post-translationally modified
3. topology of proteins determined at the ER (lumen of organelles is topological equivalent of the extracellular space)
4. protein trafficking requires membrane budding and fusion
5. endocytosis leads to recycling and degradation of proteins

Question 2

what do you need to target cargoes to a particular membrane, like the plasma membrane or the endolysosomal system?

Answer 2

signals on the proteins

Question 3

advantages of vesicular transport

Answer 3

1. proteins can move through different organelles, but only need to cross a membrane once (ER translocation)
2. sequential processing events can be compartmentalized

Question 4

challenges imposed by vesicular transport

Answer 4

1. cargoes need to be targeted to specific organelles
2. membranes can fuse and divide (energetically unfavorable) –> need a lot of ATP

Question 5

basic principles of vesicle transport

Answer 5

-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

Question 6

what are the steps to vesicular transport?

Answer 6

1. budding- requires coat proteins, membrane binding proteins, cargo receptors, and small GTP binding proteins
2. targeting- tether proteins, targeting receptors, and small GTP binding proteins
3. 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

Question 7

ER export

Answer 7

-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)

Question 8

different coats are used at different steps

Answer 8

-COPII- mediates ER budding
-COPI- between Golgi and retrograde trafficking to the ER plus trans-Golgi network
-clathrin- endocytosis

Question 9

targeting and fusion machinery

Answer 9

-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

Question 10

Golgi complex- specialized function in each cisternae

Answer 10

-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

Question 11

what are the two places the proteins can go from the Golgi?

Answer 11

1. 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
2. endo-lysosomal system

Question 12

delivery of lysosomal proteins to lysosomes

Answer 12

-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

Question 13

what do you need on lysosomal proteins?

Answer 13

-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

Question 14

lysosomal proteins are recognized by GlcNAc phosphotransferase via the signal patch

Answer 14

-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

Question 15

M6P-tagged lysosomal proteins bud off from the TGN and delivered to late endosomes

Answer 15

-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

Question 16

what do you do if you have an issue with the enzymes?

Answer 16

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

Question 17

sorting to organelles signals are the key…only lysosomal targeting involves the ER

Answer 17

-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

Question 18

signals for ER localization

Answer 18

-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

Question 19

yeast secretory pathway

Answer 19

-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

Question 20

the first secretion (Sec) mutant isolated

Answer 20

-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

Question 21

genetic screen scheme for sec mutants

Answer 21

-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

Question 22

EM determined the exact nature of secretion defect

Answer 22

-no vesicles form from the ER and the ER becomes bloated –> see accumulation of vesicles in the cytoplasm that can’t fuse with PM

Question 23

yeast secretory pathway is likely regulated by gene products

Answer 23

isolated several genetic factors important for membrane trafficking

Question 24

jim rothman

Answer 24

isolated proteins responsible for fusion at the plasma membrane

Question 25

N-ethlymaleimide (NEM) blocks vesicle fusion at the Golgi membrane

Answer 25

-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

Question 26

NSF required for fusion of cargo vesicles to target compartments

Answer 26

-you can fuse vesicles to Golgi in vitro -NSF is encoded by sec18, suggesting gene products do mediate fusion events

Question 27

performed Co-IP experiment with brain extract

Answer 27

-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

Question 28

purification of neuronal SNAREs from E. coli (liposome assay)

Answer 28

-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

Question 29

tetanus toxin to ask whether synaptic vesicle secretion is inhibited (SNARE specific proteases)

Answer 29

-when applied to neurons, showed that you cannot secrete neurotransmitter and showed that SNAREs mediate vesicle-membrane fusion

Question 30

bacterial toxins target the vesicle docking and fusion machinery of neurons

Answer 30

-botox- blocks excitation and don't have tight muscles in the face- affects synaptic transmission by cleaving SNARE proteins

Question 31

SNAREs are required for docking and fusion

Answer 31

-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

Question 32

specialized SNAREs mediate fusion at each trafficking step in the cell

Answer 32

confers specificity to the target membrane

Question 33

what does budding require?

Answer 33

1. coats 2. adaptors 3. membrane bending proteins

Question 34

coat proteins bud vesicles off from the membranes

Answer 34

-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

Question 35

GTP hydryolysis leads to uncoating

Answer 35

-in the case of COPII and COPI coats, GTP hydrolysis with GTPase the coats get disassembled -ATPase gets recruited to Clathrin and removes it

Question 36

what is endocytosis important for?

Answer 36

nutrition, defense, signaling, and homeostasis

Question 37

what are the different types of endocytosis?

Answer 37

phagocytosis, macro-pinocytosis, clathrin-mediated endocytosis

Question 38

clathrin-mediated endocytosis

Answer 38

-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

Question 39

EM with yolk uptake in oocytes in the ovary of the mosquito with apparent electron-dense coat

Answer 39

a lot of yolk uptake and looked to see coats that budded on plasma membrane

Question 40

Barbara Pearse purified clathrin-coated vesicles from the brain and looked at it with negatively stained vesicles

Answer 40

see coat on vesicles and named it clathrin

Question 41

familial hypercholesterolemia: high cholesterol level in the blood

Answer 41

-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

Question 42

adaptor protein complex binds clathrin and cargoes

Answer 42

-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

Question 43

the endocytic pathway leads to degradation or recycling

Answer 43

-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

Question 44

endosomes can be matured into late endosomes to make multi-vesicular bodies

Answer 44

buds membrane into the lumen or the organelle or endosomes --> making interlumenal vesicles

Question 45

ESCRT proteins bud vesicles into the lumen of endosomes

Answer 45

-secreted out as exosomes or degraded by lysosomes -complex pushed membrane into the lumen of organelle