Lecture #5 - Membrane Trafficking #2 Flashcards
Goal of membrane trafficking
Membrane trafficking sending proteins to the plasma membrane OR the endolysosomal system
- IF going to the memebrane = proteins will be secreted or transmemebrane proteins
ER –> Golgi –> Plasma membrane OR the endolysosomal System
ALL membrane trafficking has:
1. Porteins are co-translationally translocated to the ER
2. Protein is co-translationally and post-translationally modified
3. Topology of prteins is determined at the ER
What is required for protein trafficking to the membrane and what is the effect of endocytosis
Protein trafficking requires membrane budding and fusion
Endocytosis (endosome) leads to recycling and degradation of proteins
Pros of membrane traficking
Membrane trafficking is convinient and beneficial to compartmentalize things:
1. Can have all protein syntehsis machinery near ER (ER acts as a factory to make proteins)
2. After ER can send protein to the golgi –> sends proten to location
3. Compartemilization makes places where you can recycle things and degrade things (because of endosomes)
4. Proteins can move through different organelles but only ned to cross a membrane once (Cross membrane in ER translocation)
5. Sequential processing events can be compartmentalized
Challenges with membrane trafficking/vescile transport
- Need to target cargo to specific organelles/take crago to specific membrane (need signals on proteins to do things)
- Ex. Go to PM or endolysosomal system
- Need to divide and fuse vesciles from compartments (membranes need to fuse and divide)
Fusion and divison of membranes
Membrane traficking/vescile transport requires membrane fusion and division which is energetically unfavorable
Lots of ATP made in cell is used in membrane transport
- Example - when have less O2 going to brain membrane trafficking stops right away
Basic Principles of Vesicular transport
- Cargo moves through multiple compartments BUT only crosses a membrane once
- Membrane topology is always conserved
- Once you protein topology is detemrined at the first compartment the topology is conserved (Ex. sqaure is always in the cytoplasm)
- Have compartments for vescile transport
Compartments in vescile transport
For vescile transport have compartments:
1. Donor membrane/compartment
- At the donor = Need to concentrate cargo into the vescile that is budded off from the compartment –> vescile is then targeted to the next compartment
2. Target membrane/compartment (vescile will fuse to a second membrane)
Example – Vesicle budidng off of ER (donor) going to Golgi (target)
Steps in vesicular transport
- Budding –> uses Coat proteins + membrane bending proteins + cargo receptors + small GTP binding proteins
- Targeting –> uses Tethers proteins + targeting receptors + Small GTP binding Porteins
- Fusion –> uses Fusion proteins + disaasmbly factors
What does budinhg require
- Coats
- Adapters
- membrane bending proteins
Themes in Vesicular transport
Cargo recruited by specific cytoplasmic signals
Coats help deform the membrane
Targeting machinery is also including in budding vescile
Uncoating occurs after budding
Vescile transport (ER –> Golgi) - Overall process
Have soluble protein/ligand and receptor in the donor compartment (ligand binds to the receptors on ER membrane) –> Ligand and receptor gets concentrated in donor compartment –> coat proteins help with budding –> when vescile starts to bud off coat porteins help incprpoate other proteins into the vescile –> Vescile budds off from donor compartment –> vescile goes to acceptor compartment –> tethering components and GTPase on the vescile gets tethered onto the target membrane –> V-Snares and T-Snare engases which allows you to fuse the vesicles to a specific location
- ER membrane = donor compartment
Function of coat proteins
Coat proteins assist the mebrame bending events
- Help with budding (bud vesciles off of membranes)
- When vescile starts to bud off coat porteins help incprpoate other proteins into the vescile (Ex. V snare proteins and Rab GTPase and tetehring protein)
Types of coat proteins
COP2 - At ER
COP1 - At golgo
Clathrin - At plasma membrane and trans golgi complex
COP2 vescile formation
Overall – concetrate cargoes by adapters
- For budding needs to concentrate cargoes
Process - Once cargo is assmebled –> Adapters bind to cargo and recruits the coat to membrane –> recruiting coat promotes budding –> evnetually membrane gets cut by protein –> NOW have a vescile that is released to the membrane
- Traget machinery is also included in the budding vescile
- Have exit signal on cargo receptor and exit signal on the soluble cargo protein
Folded vs. misfoloed proteins in COP2 vescile formation
Only properly folded and assembled cargo is incorpoated into Cop2 vesciles
Chaparones bind to unfoloded or misfolded proteins
Function of tethering proteins
Tethering proteins and GTPase in vescile getting tethered to the target memebrane makes specificity for targeting that specific target membrane
Example ER export how membrane budding actually happens
Have Soluble proteins with exit siganl and transmebrane receptors with exit signal –> soluble protein binds to transmebrane receotors on the ER –> Receptors in ER membrane (receptors are now bound to the solble crago) binds to adapters –> adoaters bind to coat proteins –> generate vesicles through addition of other proteins –> once vescie is made GTP hydrolysis happens and the coat disassembles
- Soluble protein leaving ER AND receptor BOTH have an exit signal
- Coat proteins = don’t bind to receotors directly (bind to the adapters that are bound to the receptors)
Different coat proteins
Different coat proteins are used for different steps in membarne transport:
1. ER to Golgi (ER budding) - uses COP2
2. Golgi membranes and retrograding traficking back to ER - Uses COP1 to make vesicle
3. Transgolgo network - uses COP1 or Clathrin
4. Endocytosies = reuqires Clathrin
Whee does coat protein bind
Coat protein (ex. clathrin) is NOT binding to cargo or membrane directeltly
Adapters bind to the receptors/crago complex and coat protein (bind to membrane and cargoes)
Where does adapter protein bind
Adapter protein complex binds clathrin (coat) and cargoes (cargo bound to receptor)
Have multiple motifs -
- Example motif –> Tyrosine motif (THIS is the motif that adaptor proteins bind to on LDL receptor
- If have one motif on cytoplasmic tail on the proteins then they can be sorted into clathrin coated pits (protein can be sorted into coated pits)
Example adapter protein = AP2 (works at plasma membrane)
Different types of Adapter proteins
AP1 and AP3 = binds to clathrin at the trans golgi network (calthrin works at trans golgi network)
Binding of AP1 (adater protein 1) and AP3 (Adpater protein 3) = determine where the crago is sent
- IF cargo doesn’t have binding to AP1 then it is sorted to AP3 and goes to certain location
- Binding to AP1 sends protein to basolateral side ; binding AP3 sends protein to apical side (Affinity for AP1 decides what protein goes to what compartment in cell)
- Adapter proteins bind the same sequence BUT the affinity of binding decides where the proteins go
Where does AP3 and AP1 work
Works in endosomes to sort proteins AND work at the trans golgi network
AP2 = works at the plasma membrane
What happens after budding
After budding have vesicles that is release from membrane –> have GTP/ATP hydrolysis and then coat disasembley
COP2 and COP1 coats use GTP hydrolysis of GTPase to disasemble the coat
- Sec23 is the GAP (GTPase activating protein) for Sar1
- Phosphorylation of COP2 subunits at the target membrane also contrubutes to uncoating
Clathrin uses ATPase that gets recruited to clathrin to disasemble the coat
What does targeting require
Targeting requires V-SNARES and T-SNARES
V-Snare (vesicular Snare) = on the vesicle
T-Snare (target Snare) = on target compartment
V-SNARE and T-SNARE make a specific pair that can mediate fusion
- IF they are not the right partners then they can’t mediate fusion = Confers specificity for fusion events
Function of Snares
SNAREs confer specificity and mediate docking and fusionof vesciles to target membrane
Specificity - only certain V-SNAREs and T-SNARES can interaction
Specilized Snares
Different membrane fusion events use diffrent SNAREs AND use different Rab GTPase/receptors for tethering
- Specialized SNAREs mediate fusion at each trafficking step in the cell
BOTH Snares and Rab GTPase confer specificity of vesicles to target membranes
What disassembles SNAREs
NSF ATPase dissembles SNARE pairs
After Fusion with SNARE assembly –> Cis-Snare Complex needs to be dissassembled
- Cis-Snare complex = one of the tightest complex found in cell = requires energy (ATP) to unwind/disassemble
- NSF = ATPase that unwinds the Cis-SNARE complex –> THEN can recycle Snare protein for next vesicle fusion event
Tether proteins
Tether proteins bind Rab GTPAse
- Rab GTPase binds to Rab effector (Rab effector = tethering protein) –> binding of Rab to tether protin mediates tethering
Function - targets vesicles to certain locaton
- Each vesicle has specific Rab GTPase associated with them and have Rab receptor for tethering proteins on target membrane that binds to Rab = send certain vesciles to target membrae
- Rab receptor = addresss to send certain vesicles to the target membrane
What happens once Rab binds to receptor
Once Rab binds to receptor = SNARE proteins are associated and mediate fusion events
- Only fuse once Rab binds to receptor
Golgi - Overall
Golgi = shipping center –> sends particular protein to certain locations (sort at begining and end)
- All the proteins are sorted at the Trans golgi network and are sent to the traget membrane
After golgi the protein can go to Plasma membrane or endolysosomal system
Image - See Golgi Stacks (each stack has different function)
Additional functions of golgi
Different Stacks have diferent functions
Main function = modify proteins –> As protein comes form ER the proteins are sorted in golgi THEN the glycosylation on the proteins can be further processed in golgi
Example functions - Sorting of proteins and lipids + protolycan assembly + Processing of N-linked oligio saccarides + o-glycosytlation (Ser and Thr residues) + Phosphorylation of sugars + Proteolytic procesisng + Tyrosine sulfanation
Sugar modifications in golgi
- Galactose can be added + NANA can be added to glycosylated proteins
- Important for distiction of self to foreign materials so body knows proteins are not foriegn (if have issue with this pathway your immune system makes auto-AB against proteins that are secreted)
- O-glycsylation –> Added on OH on Ser and Thr
- Important for adhesion molecules
Where do proteins go after golgi
Two places proteins can go from the golgi = Plasma membrane or endolysomal pathway
- Plasma membrane (defaut oathway)
- IF there is no other modification that happens on the protein then protein goes to the PM
- Get secteroy vesicle or PM protein
- Can send proteins to the endolysosomal pathway (proteins go to lysosomes)
Options for proteins at the plasma memebrane
At plasma mebrane can have regulated secreion or constitutive secretion -> have exocytosis at the plasma membrane
- Ex – Insulin has regulated secretion –> Have external signals that says when to release ptotein
- IF have no regulation = Have exocytosis at the plasma membrane (secretroy vescile) = constitutive secrtory pathway
Constitutive secretroy pathway = default pathway
Function of lysosomes
Lysosomes = Degrades proteins and lipids + Lysosomes are also a signlaing hub
- Lysosmes has acid hydrolases to degrade things
Need to send many enzymes to lysosmes
- Membrane trafficking from ER to the golgi = way to send proteins to the lysosomes
IF you can’t send proteins to the lysosomes = get disease (Ex. Gauchers ; Tay Saches ; Mucopolipidosies)
How do you send proteins to the lysosomes
Overall – done by further modifying sugars on proteins
- Lysosomal proteins are tagged with mannose-6-Phosphate
Process - Glycosylated protein from the ER goes to golgi –> GlcNac-Phosphate is added on the mannose surgar on protein you want to target to lysosome by phosphotransferase (added to the mannose 6 position) –> THEN GlcNac is removed by phosphodiestrase –> NOW only mannose-6 phosphate is on the sugar molecule
What happens to proteins that have mannose-6-phosphate
At the Trans golgo network have Mannose-6-Phsophate tagged proteins –> proteins bind to M6P receptrs on golgi –> protein is sorted at the Trans golgi network by clathrin and adpater complex (AP1) –> buds whatever is bound to M6P receotor –> send protein in vesicle to the late endosome through the endolysomal system (remove phosphate in late endosome and the lysosomal protein dissociated from the M6P receptor due to low pH) –> through maturaturation and fusion vesicle the lysomal protein gets delivered to the lyososme and M6P receptors are recylced to the Trans golgi network
Are all glycosylated proteins tagged with Mannose-6-phosphate
NOT all glycosylated proteins get M6P because GlcNac Phosphotransferase has specificity to the structure of proteins that it will modify
Only sepecifc proteins can bind to GlcNac phosphotranserase = GlcNac phosphate is added to the mannose 6th positon = get the intermediate protein and THEN phosphodiestrase removes GlcNAc and have the phosphate added to mannose 6 = send the protein to the lysosomes
Proteins tagged with M6P escaping pathway
Even if you tag a protein with M6P it can escape from the golgi and goes to default (sectory) pathway
BUT will ahve some M6P receptors on the cell surface – because have M6P recpetor on surface you can have exocytosis of the protein BUT then the protein binds to M6P in cell membrane –> have endocytosis –> early endosome –> late endosoe –> goes to the lysosome
Use of M6P on cell mambrane
If you have problem with lysomal enzyme = do eynzyme replacement therapy
Have a M6P tagged enzyme that is added into the extracellular space –> enzyme will bind to M6P receptor on cell membrane –> vesicle will go through endocutosis and maturation of endosomes –> enzymes go to lysomses –> alleviates symptoms
How do you send proteins to different organelles in the cell
Mitocondria + peroxisomes + Nucleus = don’t need ER golgi membrane traficking pathway + all have post translational translocation
- Proteins are made in the cytoplasm and then get targeted to the organelles
Have different siganls for different locations (signals are often lysine rich)
Retaining protein in ER or Golgi
Retaining a protein in ER or golgi requires signals on the proteins
- Signal = ER retention/ER retrieval signal
Example:
1. Soluble proteins that = function in the ER have KDEL sequence (allows you to keep proteins in ER)
2. Transmembrane protein in ER = Have signal on cytoplasmic side (have Dilysine motif at end of C terminus tail)
- Ex. Sec61 has signal and will stay in ER
Discovery of proteins essential in teh secrtory pathway
Used yeast genetic screen and isolated a mutants with inhibited secretion of soluble proteins
Image – Left is normal ; right is temperature sensative mutant that can’t divide but can syntehsize proteins
- Mutant at restrictuve temperature could not secrete proteins (accuumulation of vesciles inside the cytoplasm) –> THINK that because the cells can’t secrete proteins they will be heavier
What did they do after finding the temperature sensative mutant
After they found the mutanet –> spun the cells in sucrose gradient and found the heavier cells go to a lower band in sucrose gradient
Did random mutagensis again –> spun cells –> see heavier cells –> isolate teh cells
End – mapped 188 strains onto 23 different genes
What did they do after mapping the genes
Characterize what pathways the gene products may be acting in using Electron microscopy
Left - Left Sec mutants with no vesciles from ER and the ER is broader = can guess what gene product is doing (Maybe the gene product is making vesciles from ER)
Right – Sec mutant with extra vesciles ; inability to fuse with plasma membrane
Using this they can map where the gene products are acting
Results of biochem experiments to look a what the gene products do
Isolated the proteins that are responsible for fusion vesicles at the plasma membrane –> Found that the SNARE proteins form a complex
Found using a drug that blocks fusion of vesicles to golgi (NEM)
- When add NEM to cell –> have many vesicles associated with the golgi BUT can’t actually do fusion
- Used NEM to purify the proteins that are required for the process/act in fusion
Using NEM they found NEM sensitive factor (NSF) = cytosolic and can be extracted fro purified golgi membrane by washing off NEM and the addition of ATP (found NSF is component in pathway)
Steps once have NSF
Once have NSF –> do in vitro assay where add NSF to purified golgi and vescile that previously was not able to be fused
Found – When add NSF the vesicles can now fuse to the Golgi
- Found NSF was encoded by sec18 in yeast –> suggests the gene product of Sec do mediate fusion
Issue – didn’t know if NSF mediate fusion itself OR is just involved
Nomenclature
Really just for reference
Just know that V-Snare is on the vesicle ; T-Snare is on the target membrane
Liposome function Assay
Once know 3 compoents –> VAMP + SNAP-25 + Synthatic –> can make recombinatnt proteins in bacteria and purify the proteins and put them into liposomes and see if the liposomes fuse
Had 1 compoent in 1 vescile (V-SNARE donor vesicle) and 1 compoent in another vescile (T-SNARE acceptor vescile) –> Ask if the vesciles fuse
- Found that if the components are on different sides of membrane (on different vesicles) they mediate fusion
- BUT if both vesciles have V-SNARE there is no fusion ; of both vesciles have T-SNARE there is no fusion –> means you nee V-SNARE on one vesicle and T-SNARE on the other side
END - showed that SNAREs are sufficinet for fusion of liposomes (SNARE mediates fusion NOT NSF)
Liposome
Liposomes = vesciles made of lipids and detergents
2nd experiment to show that SNARE mediated fusion
Done using toxins/proteases
Used tetanus Toxin to ask if the synaptoc vescile secertion is inhibited
- When add tetnus to nuerons –> NOW they can’t secrete nuerotransmitters = shows that SNARE mediates vesciles fusing to the membrane
END - Found that tetnus blcoked nueronal secretion
Toxins similar to tetnus
There are other toxins that do similar things to tetnus
- Bacteria toxins target the vesicle docking and fusion machinery of nuerons
The light chain of clostridial nuerotoxins clave specifc nueronal SNARE proteins
Botox = works by blocking the excicitation by affecting synaptic ransmission upon cleavge of SNARE proteins = muscles will relax (ex in face)
Types of endocytosis
ALL use diffrent mechanisms
- Phagocytosis
- Macro-pinocytosis
- Clathrin mediated endocytosis (best known endocytosis pathway)
- Caveolin mediated endocytsosis
- Pinocytosis not meadited by clathrin or caveolin (clathrin indepedent)
Clathrin mediated endocytosis
Budding = similar to COP1/COP2 mediated budding
Budidng - cargo is bound to receptor –> adapter to bind to receptor –> adaptors recuit clathrin to site (coat assembly and coat selection)–> recruitment of clatherin promotes the budding of membrane (bud formatino)–> eventually make vescile which is cut from the membrane to get final vescile (vescile formation) –> uncoating
How does clathrin coat disasselble
ATPase comes in and disassembles the machinery –> clathrin coat and adpater disasscoaites from the vesicle –> NOW have free vescile that is transproted to the endosomes
How did we discover clathrin mediated endocytosis
Discovered in EM
Looked at yolk uptake in oocytes in the overy of masquito with apparent electron dense coat
- Oocyetes = profesional endocytic cell
EM saw there are coat on membrane that is budding on plasma membrane (knew endocytosis BUT didn’t know what the coat on membrane was)
How are cargoes selected for internalization
They didn’t know how clathrin machinery sorts proteins into vesciles at Plasma membrane
They used pateints with familial hypercholstremia (didn’t know why they had so much cholestral in their blood)
Sequenced the LDL recpetor –> found a mutation in the cytoplasmic tail of the receptor that leads to missorting of LDL recpetors from coated pits
Experiment after found mutation on LDL receptpr
After found mutation – isolated fibroblasts from pateints and looked at endocytosis of recpetors using Antibody for LDL
Normal fibroblasts have endocytosis that happens normally –> LDL is sorted/concetrated into coated pits on surface and they get endocytosed (internalized) normally
Pateint fibroblasts –> Endocytosis happens with clathrin vescile BUT the LDL is not concetrated into the pit
- LDL is just on the PLasma membrane and the antibody for LDL is NOT internalized
Showed that the mutation in the cytoplasmic tail is important for sorting proteins into clathrin coated pits AND that if you mutate the tail you can’t internalize LDL
What binds to motif on cytoplasmic tail
Clathrin doesn’t bind the Try motif ; adpater proteins does bind to this motif
Tyrosine motif = motif that adaptor proteins bind to on LDL receptor
If have one motif on cytoplasmic tail on the proteins then they can be sorted into clathrin coated pits
Important proerty of endosomes and lysosomes
Endosomes and lysosmes are acidic –> allows signaling molecules and nutrients to dissociate from receptors
MEANS It is important to get the protein to endosomes or lysomes because the endosomes and lysosomes are acidic
- pH of exracellular = 7.4
- pH of endosomes = 6
- pH of lysomes = 5 (sometimes 4.5)
Use of acidic envirnment in endosomes and lysosomes
In the acidic environment of endosomes or lysomes the ligand and receptor are dissociated = now the receotors can go to the plasma membrane to be recyled and the ligand can be sent to next compartment and can used for redistrbution to cell membrame/organelle membrane
Example LDL in endolysosomal pathway
Example - LDL and LDL receptor sort into the clathrin vesicle –> Vesicle goes to endosomes –> at the early endosome the ligand LDL dissociates from LDL receptor –> LDL receptor goes to palsma membrane to be recyled and ligand LDL goes to the lysomes –> at the lysomes the portein in LDL is degraded and cholstril stays (cholstrol is in LDL complex) –> proteins NPC1 and NPC 2 pick up the free cholestral and put it into the lysomal membrane and resitrbute the chrolestrol to eh ER and the plasma membrane
- Dissociation of ligand and receptors requires acidic pH
Intraluminal vesicles
Membrane proteins are internalized –> sent to endosomes –> sorted into the intraluminal vesciles
Other budding can bud membrane to cytopalsm BUT THIS buds the membrane into the lumen of endosomes –> makes interluminal vesicles
- Intraluminal vesicles are made by a different set of proteins
Maturation of endosomes
At endosomes – endosomes can be matured into late endosomes to make multi vesicle bodies
- THIS IS a membrane budding event that is different from other budding
Other budding can bud membrane to cytopalsm BUT THIS buds the membrane into the lumen of endosomes –> makes interluminal vesicles
ESCRT proteins
ESCRT proteins bud vescules into the lumen of endosomes
- ESCRT proteins sort proteins and bud them into interluminal veciles –> they (interluminal vesicles?) can be secreted at exosomes or they can be degraded by the lysosomes
Complex pushes the membrane into the lumen of organelles = components are different from clathrin components (push membrane inward)
Same protein used at Plama membrane for generating exosomes
Answer C
KNOW it is not A, E, or D
Default of proteins = leave the ER
Part A
Answer - ER
Right when ahve signal sequece have transolation to ER
- ER is the first destination for protens going to plamsa membrane or golgo or endosome (where goes after ER depends on their signals)
- Mit ; peroxsisomes ; Nucleus = goes to organelles after translaton
- Once protein is in the ER the NLS is usless
Part B
Anser - ER (same logic as A)
Once go to ER the proxisomeal signal doesnt matter (because one in teh ER the peroxisome machinery can’t see the signal)
Part C
Answer - ER
ER retrival signal makes the protein go from the golg back to ER
- KDEL signal on soluble proteon or teh Dilys signal on trasnmemebrane proteins = get sent back to ER
- When have retrival signal you don’t go through full golgi steps = won’t go to plasma membrane
Part D and E
D = Golgi and stay there
- ER signal wins over the mitocidniral pre sequnece so ther proteins goes to ER lumen and then because have golgi localization so they get stuck in golgi
E = lysomes (M6P addition sends proteins to lysosome)
Repeate the experiment and stain for epitipe B
Answer - A
You analyze a trucnation mutant lacking teh C terminal 10 amino acids using an AB for epitope A
Answer - A and C
