Lecture #5 - Membrane Trafficking #2

Question 1

Goal of membrane trafficking

Answer 1

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

Question 2

What is required for protein trafficking to the membrane and what is the effect of endocytosis

Answer 2

Protein trafficking requires membrane budding and fusion

Endocytosis (endosome) leads to recycling and degradation of proteins

Question 3

Pros of membrane traficking

Answer 3

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

Question 4

Challenges with membrane trafficking/vescile transport

Answer 4

1. 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
2. Need to divide and fuse vesciles from compartments (membranes need to fuse and divide)

Question 5

Fusion and divison of membranes

Answer 5

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

Question 6

Basic Principles of Vesicular transport

Answer 6

1. Cargo moves through multiple compartments BUT only crosses a membrane once
2. 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)
3. Have compartments for vescile transport

Question 7

Compartments in vescile transport

Answer 7

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)

Question 8

Steps in vesicular transport

Answer 8

1. Budding –> uses Coat proteins + membrane bending proteins + cargo receptors + small GTP binding proteins
2. Targeting –> uses Tethers proteins + targeting receptors + Small GTP binding Porteins
3. Fusion –> uses Fusion proteins + disaasmbly factors

Question 9

What does budinhg require

Answer 9

1. Coats
2. Adapters
3. membrane bending proteins

Question 10

Themes in Vesicular transport

Answer 10

Cargo recruited by specific cytoplasmic signals

Coats help deform the membrane

Targeting machinery is also including in budding vescile

Uncoating occurs after budding

Question 11

Vescile transport (ER –> Golgi) - Overall process

Answer 11

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

Question 12

Function of coat proteins

Answer 12

Coat proteins assist the mebrame bending events

1. Help with budding (bud vesciles off of membranes)
2. 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)

Question 13

Types of coat proteins

Answer 13

COP2 - At ER
COP1 - At golgo
Clathrin - At plasma membrane and trans golgi complex

Question 14

COP2 vescile formation

Answer 14

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

Question 15

Folded vs. misfoloed proteins in COP2 vescile formation

Answer 15

Only properly folded and assembled cargo is incorpoated into Cop2 vesciles

Chaparones bind to unfoloded or misfolded proteins

Question 16

Function of tethering proteins

Answer 16

Tethering proteins and GTPase in vescile getting tethered to the target memebrane makes specificity for targeting that specific target membrane

Question 17

Example ER export how membrane budding actually happens

Answer 17

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)

Question 18

Different coat proteins

Answer 18

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

Question 19

Whee does coat protein bind

Answer 19

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)

Question 20

Where does adapter protein bind

Answer 20

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)

Question 21

Different types of Adapter proteins

Answer 21

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

Question 22

Where does AP3 and AP1 work

Answer 22

Works in endosomes to sort proteins AND work at the trans golgi network

AP2 = works at the plasma membrane

Question 23

What happens after budding

Answer 23

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

Question 24

What does targeting require

Answer 24

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

Question 25

Function of Snares

Answer 25

SNAREs confer specificity and mediate docking and fusionof vesciles to target membrane Specificity - only certain V-SNAREs and T-SNARES can interaction

Question 26

Specilized Snares

Answer 26

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

Question 27

What disassembles SNAREs

Answer 27

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

Question 28

Tether proteins

Answer 28

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

Question 29

What happens once Rab binds to receptor

Answer 29

Once Rab binds to receptor = SNARE proteins are associated and mediate fusion events - Only fuse once Rab binds to receptor

Question 30

Golgi - Overall

Answer 30

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)

Question 31

Additional functions of golgi

Answer 31

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

Question 32

Sugar modifications in golgi

Answer 32

1. 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) 2. O-glycsylation --> Added on OH on Ser and Thr - Important for adhesion molecules

Question 33

Where do proteins go after golgi

Answer 33

Two places proteins can go from the golgi = Plasma membrane or endolysomal pathway 1. 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 2. Can send proteins to the endolysosomal pathway (proteins go to lysosomes)

Question 34

Options for proteins at the plasma memebrane

Answer 34

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

Question 35

Function of lysosomes

Answer 35

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)

Question 36

How do you send proteins to the lysosomes

Answer 36

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

Question 37

What happens to proteins that have mannose-6-phosphate

Answer 37

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

Question 38

Are all glycosylated proteins tagged with Mannose-6-phosphate

Answer 38

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

Question 39

Proteins tagged with M6P escaping pathway

Answer 39

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

Question 40

Use of M6P on cell mambrane

Answer 40

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

Question 41

How do you send proteins to different organelles in the cell

Answer 41

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)

Question 42

Retaining protein in ER or Golgi

Answer 42

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

Question 43

Use of ER retention/ER retrieval signal

Answer 43

Even if have signal sequence the protein can go to Golgi BUT then they will go back to ER from Golgi (WONT go to plasma membrane will go to ER)

Question 44

Discovery of proteins essential in teh secrtory pathway

Answer 44

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

Question 45

What did they do after finding the temperature sensative mutant

Answer 45

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

Question 46

What did they do after mapping the genes

Answer 46

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

Question 47

What did they do after EM

Answer 47

Based on screens --> isolated several genetic factors that may be important for membrane trafficking (Know the proteins are important BUT does’t say proteins are actually involoved in the processes) Needed to still show that the proteins actually does membrane trafficking NOW need to use biochem

Question 48

Results of biochem experiments to look a what the gene products do

Answer 48

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)

Question 49

Steps once have NSF

Answer 49

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

Question 50

Step after know NSF is involved in fusion

Answer 50

Do Co-Ip using NSF as bait to pool all of the components needed for fusion - Use Antibody for NSF --> when do Co-IP AB bound to NSF would bring everything that NSF binds to in a complex Things that came with NSF = NSF soluble attatched protein - Found there were 2 soluble comeponents attatched to NSF (SNAP) AND a SNAP receptor (SNARE) Once have proteins – Add trpyin to complex and sequence --> found the proteins were coded for by SNAP25 , Syntaxic, and Synaptobrevin/VAMP genes

Question 51

Nomenclature

Answer 51

Really just for reference Just know that V-Snare is on the vesicle ; T-Snare is on the target membrane

Question 52

Question once have NSF and SNARE proteins

Answer 52

Is it NSF or SNAREs that mediate fusion How did they answer question (experiments done to show that SNARE mediates fusion): 1. Liposome Function Assay 2. SNARE-specific proteases

Question 53

Liposome function Assay

Answer 53

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)

Question 54

Liposome

Answer 54

Liposomes = vesciles made of lipids and detergents

Question 55

2nd experiment to show that SNARE mediated fusion

Answer 55

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

Question 56

Tetanus Toxin

Answer 56

Tetnus Toxin = targets SNARE proteins - Tetnus cleaves the vesicular SNARE (V-SNARE) synaptobrevin

Question 57

Toxins similar to tetnus

Answer 57

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)

Question 58

Importance of endocytosis

Answer 58

1. Important for homeostasis 2. Important for nutrient uptake 3. Important for defense 4. Important for signlaing

Question 59

Types of endocytosis

Answer 59

ALL use diffrent mechanisms 1. Phagocytosis 2. Macro-pinocytosis 3. Clathrin mediated endocytosis (best known endocytosis pathway) 4. Caveolin mediated endocytsosis 5. Pinocytosis not meadited by clathrin or caveolin (clathrin indepedent)

Question 60

Clathrin mediated endocytosis

Answer 60

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

Question 61

How does clathrin coat disasselble

Answer 61

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

Question 62

How did we discover clathrin mediated endocytosis

Answer 62

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)

Question 63

Discovery of clathrin after EM

Answer 63

After EM – coated vesicles were purified --> looked at cryo EM --> saw coat on vesciles and named the coat proteins clathrin

Question 64

How are cargoes selected for internalization

Answer 64

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

Question 65

Experiment after found mutation on LDL receptpr

Answer 65

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

Question 66

What binds to motif on cytoplasmic tail

Answer 66

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

Question 67

Overall proteins involoved in clathrin mediated endocytosis

Answer 67

NOW know there are 60 proteins recuirted to the site of endocytosis at different times in clathrin mediated endocytosis (during crago recruitment or adpater recruitment or vescile budidng) - ~60 proteins involoved in clathrin mediated endocytosis - Proteisn are regulated by different mechansims in diferent cells

Question 68

Function of endocytic pathway

Answer 68

The endocytic pathway leads to protein recyling or degradtion Once endocytosis happens the vesicles go to endosomes --> from there some of the crago is reczyled to plasma membrane OR some go to endosoems OR endolysomal system

Question 69

Important proerty of endosomes and lysosomes

Answer 69

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)

Question 70

Use of acidic envirnment in endosomes and lysosomes

Answer 70

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

Question 71

Example LDL in endolysosomal pathway

Answer 71

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

Question 72

Intraluminal vesicles

Answer 72

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

Question 73

Maturation of endosomes

Answer 73

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

Question 74

ESCRT proteins

Answer 74

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

Question 75

Use of ESCRT system

Answer 75

System has been hijacked by viruses to make capsid Important for virology + protein homeostasis + exosome secretion

Question 76

Endocytosis importence

Answer 76

Overall – endocytosis is important for many things Send vesicles to endosomes and proteins go to different locations from there

Question 77

Is export signal essential

Answer 77

Export signal is NOT essential just makes export more efficient You can get to he next compartment without the export signal

Question 78

Where can proteins made in the ER go

Answer 78

Proteins made in ER can be mebrane proteins or screted or go to golgi or endolysomal ssystem

Question 79

Answer 79

Answer C KNOW it is not A, E, or D Default of proteins = leave the ER

Question 80

Part A

Answer 80

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

Question 81

Part B

Answer 81

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)

Question 82

Part C

Answer 82

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

Question 83

Part D and E

Answer 83

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)

Question 84

Question 85

Answer 85

Question 86

Repeate the experiment and stain for epitipe B

Answer 86

Answer - A

Question 87

You analyze a trucnation mutant lacking teh C terminal 10 amino acids using an AB for epitope A

Answer 87

Answer - A and C