Lecture #4 - Membrane Trafficking Flashcards
Overview of membrane trafficking pathway
Overall – proteins go from the ER –> golgi –> proteins goes to a secretory vesiccle or early endosome –> plasma membrane or enodolysosomal system
30% of human proteins follow this path
50% of drugs traget plasma membrane proteins = important to understand the pathway
MAJOR TAKE WAYS from lectures
- Proteins are co-translationally translocated to the ER
- Topology of the proteins is determined at the ER
- Once topology is determined the topology is conserved across membrane trafficking (means if facing the lumen of the ER then you will face the extrcellular space)
- Proteins are co- and post tranlsationally modified
- Protein traficking requires membrane budding and fusion (requires energy)
- Endocytosis leads to recycling and degradation of proteins
Topological equivilent of lumen of organelles
The lumen of organelles is the topological equivilent of the extracellular space
Types of proteins that can be secreted to the plasma membrane
- Soluble proteins (inside lumen they are not attatched to a membrane)
- When soluble proteins are scereted –> they go to the extracellular space and diffsuse
- Transmembrane proteins (aka membrane anchored proteins)
- Anchored to the membrane of the vesciles after release from golgi –> dleivered to plasma membrane –> act as a transmembrane protein once at the plasma membrane (stay at membrane)
What are soluble/membrane proteins loaded into
Soluble/membrane proteins (cargo) = loaded into vesciles
From the gologi the proteins bud off inside vesciecles (with the secretory proteins inside or the cell mebrane protein imdeded in mebrane of the vescile) –> vesicles will go to the plasma membrane ad willrealse the secrgted protein or embde the cell membrane protein in teh cell membrane
How was the secretory pathway discovered
Discoerved by Jamison and palade
Discovered membrane trafficking through electron microscopy studies on pancreatic acinar cells that secrete digestive eznymes
From EM – they found vesicle (called ‘granuals’) that accumulate in the cytoplasm (had granials with digestive enzymes)
- In the exocrine panecrus cells - >85% of proteins synthesized go into secretaory granials
Experiment (to see how granuals are made and how they are secreted)
Doing pulse chase to follow protein synthesis to secretion by EM
Use radiolabled Leucine (added Leu for 3 minutes to slice of pancreus) –> new proteins made will incorporate the radiolabled leucine –> chase in unlabled leucine at different times –> fix overlay with phtographic emulsion to detect radioactivoty –> process samples for Electron microscopy to locate radiolabled compartemts
- Looked at different time points after application of Leucine
Experiment (to see how granuals are made and how they are secreted) - results
0 minutes after application (right after applied leucine) –> the newly synthesized proteins that incorporated leucine are found in the ER membrane
- ER membrane is the only place the proteins are = suggest the ER is where the protein is made
After 17 minutes –> radioactivity is in the golgi
- Means from the ER the proteins go to the Golgi
After 37 minutes –> the radioactivity goes into the granials (granuales are close to the Golgi and far from the plasma membrane)
After 117 minutes –> the radioactivity is in the granules that are now close to the plasma membrane + the lumen (extracellular space) of the organ has radioactive material = suggests that the proteins are secreted into the lumen
End pathway that the Jamieson experiment came up with
OVERALL – suggests there’s a membrane trafficing pathway that goes from the ER to the golgo to granilas THEN the protein is scerteed to outside of the cell (THIS IS THE MEMBRANE TRAFICKING PATHWAY model that the expeirment came up with)
Preidctions from the membrane trafficking model (model has several predictions)
- Cargo is loaded at the ER
- Proteins are requried for traficking
- There is budding and fusion of vesciles from the ER to the Golgi and from the gogli to the plasma mebrane (this requries energy because membrane-membrane fusions is not energetically favorable = means that fusion requires proteins)
- Cargos are sorted and delivered to the right targets (Protein has to go from ER to the golgi them the plasma membrame)
How are crago synthesized and loaded at the ER?
Things that are required for protein to be synthesized and loaded at the ER:
1. Signal seqeunce on nascant peptdes
2. Signal recognition Particle (SRP) and signal recognition particle receptor needed for ribosome tranlsatiion to ER
- SRP receptor = on ER membrane
- SRP and its receptor is needed to bring the proteon/ribsome/mRNA complex to the ER
3. Transclocon (sec61) to channel the polypetides into ER lumen
Signal sequence (overall)
Signal sequence = 16-30 Amino acids long (sequence needs to span the transmembrane length)
Gunter Blobel
Discovered the intrinsic signals of proteins that governs that transport and localization in the cell (discovered protein translocation into the ER)
Types of ribosomes
2 types of ribsomes:
1. Soluble cytosolic ribsomes (ribosomes in the cytoplasm)
2. Ribsomes that were asscoiated with the ER
Translation on the types of ribosomes
Have selective translation of specific mRNA on cytosolic ribsomes and ER-associated ribsomes
- Know ribosome tranlsate mRNA
Cytoplsmic ribosomes can translate proteins that are normaly in the cytoplasm (ex. ferritin)
- ER ribosomes normally makes proteins that are secreted
ER ribosomes translate mRNA that codes for proteins that get secreted the extrellular space (lumen) (Ex. Albumin)
Know Nascant peptides ca be released into ER
How do we know that the different ribosomes translate different mRNA
Experiment – looking at immunoglobulin light chains that are secreted from cells
Have immunoglobulin made from free ribosomes (normally make proteins that stay in cytoplasm) -> proteins run at a larger size on western
Make the same immunoglobulin protein with ribosomes on the ER membrane (make secreted proteins) = the size of the protein is smaller
END – ribosomes in a tube (free ribsomes) made bigger proteins Vs. Ribsomes that are on ER membarne made smaller proetin
What did they predict based on immunoglobulin western
Based on western – made Signal sequence hypothesis
Signal sequence hypothesis = N terminal of nascant petides have a sequence that is recognized for ER targeting
- Based on fact that the protein made from ribosomes on the ER = smaller = think the protein is cut inside the ER
- Sequence is cleaved off after translation (part of teh protein is cut off inside the ER)
Is the signal sequence hypothesis true
When tested hypothesis they found it was true
MEANS – that when the protein is in the ER – part of the protein is cut off (part that is cut off = signal sequence) –> proteins made from ER ribosomes are smaller
- Signal sequence = signal to send the polypetide to the ER lumen
Signal sequence function
Signal sequence function – directs proteins into the secretory pathway
Signal sequence = cells cue for the protein to be made in the ER
Signal sequence common features
- String of hydrophobic Amino Acid residues (16-30 AA)
- Basic residues next to signal sequence (next to hydrophobic core)
- Cleavage site for signal peptidase
Signal sequence hydrophobic Amino Acids
Signal sequence hydrophic residues = spans the membrane
Hydrophicbic core = true signal sequence
Signal sequence – dictates if the peptide will go into the ER lumen or not
Signal sequence basic residues
Basic residues next to signal sequence (next to hydrophobic core)
- BAsic resudens = Arg; Lys ; His
Function - Basic residues = dictate the orientation that the peptide goes into the ER
Signal sequence cleavage site
Cleavage site for signal peptidase
Most signla sequneces are cleaved off at Ala-X-Gly or Gly-X-Ala
After tranlocation of the peptide to ER IF have the cleavge site = protein is cut (cut off signal sequence)
- Signal sequence gets cleaved off
Exception – some signal sequences are NOT at the extreme N terminus and some signal sequences are no cleaved (not all proteins have a cleavage site)
What do all ER proteins have
NOT all proteins have a cleavage site ; not all proteins have basic residues BUT for secreted proteins you NEED the hydrophobic core (all have teh hydrophobic core)
Hydrophicbic core = true signal sequence
What happens when a signal sequence is translated
When teh ribosome translates signal sequence on the nascant peptide –> the signal sequnece (hydrophobic signal) is recognized by the signal recognition particle (SRP)
Once the SRP binds to the signal sequence translation pauses –> THEN the SRP translocates and brings the complex with ribsome/protein to SRP receotors on ER membrane (this process brings the complex to the ER membrane)
- Overall – SRP and SRP receptors are needed for the ribsomes/protein/mRNA complex to be brought to the ER
- SRP brining complex to teh ER = ‘targeting’
Once brought to the ER the Nacant peptide goes into the chanel (tranlocator Sec61) –> SRP will detatch –> translation restarts and happens in the ER
Why does a prtein/ribsome/SRP complex go to the ER
Goes to ER membrane because of teh signal sequence and SRP (SRP and the signal sequence) = show teh cell that the proptein shoudl be made in teh ER
Overview of ER translocation (bring the protein/mRNA/ribsome to the ER)
Image – Signal sequence emerge from ribsomes -> SRP binds to the signal seuqnwece which causes a pause in transaltion (‘recognition’) –> targeting for the SRP to the SRP receptor on the ER membrane –> after the Rbsome/mrNA/protein binds to the SRP receptor the whole thing can tranlate lateraly in the ER membrane (moves to the side out of tranlocasor) –> tranlation will continiue and tranlocation begins
Why does SRP bind to the Signal sequnece
Signal sequence = hydrophobic = issue because you don’t want a hydrophobic thing in the aqueous solution of the cytoplasm
Solution – SRP sees and binds to the hydrophobic residues (SRP recognizes the signal sequence) –> THIS pauses translation
What is teh SRP receptor close to
SRP receptor will be close to a protein translocator on the ER membrane
Want the peptide to go into the ER
Want the peptide to go into the ER –> have a chanel that is needed to translocate the peptide into the ER lumen
Protein that will translocate the peptide into the ER lumen = tranlocaon (aka Sec61) (light blue in image)
Experiment to find Sec61
Used genetic screens in yeast
Goal – looking for the protein that translocats the protein into the ER lumen (laready knew the signal sequence exists)
- Histadine is not an essential amino acid in yeast because they can make Histadine using His4
Process – put His4 into the ER lumen (done by putting a signal sequence onto the protein) so His4 woudl only be in the ER lumen
NOW – the yeast die unless you get them Histadine because they can’t acess the His4 to make histadine if the His4 is in the lumen
Once His4 is in the ER lumen –> give yeast Histastine and do randonmon mutagensis
After mutagensis give yeast histadiol and look for survivors
Survivors in Yeast Screen
Survivirs have mutations in His4
Mutaion could be a revertant mutation that moves His4 to teh cytoplasm
Mutations could give the translocation protein (sec61) –> becauase have teh translocation protein after mutageiss His4 can go to the cytoplasm = yeast can grow without histadine being added
Once find mutant – mapped protein to Sec61 gene and found that Sec61 is the protein chanel that translocates the proteins into the ER lumen
Sec61
Sec61 = protein chanel with large pore that sits on the ER membrane
Normally have a plug that sits in Sec61 chanel BUT when the peptide comes in the plug is displaced so the channel is open = the peptide with signal sequence can go through the channel and the rest of the protein can be translated
- Plug makes sure that nothing gets transproted from the cytoplasm to the ER lumen
What happens once the protein goes through Sec61
Once the protein goes through the channel (now have hydrophobic signal sequnce transmembrane domain in the chanel) –> Sec61 can open on side –> protein can leave Sec61 chanel and go into the ER membrane (moves laterally in the membrane and becomes the transmembrane protein that anchors in the ER protein)
- Laternal movement hapens during translation
- Ejecting protein frees the translocon
End – Sec61 opens and the peptide can escpae into the ER membrame
How is the signal sequence cleaved
Overall - have a peptidase that cleaves the signal sequence
When is the signal sequence cleaved
Signal sequence is cleaved co-translationally
- As translation hapens the signal sequence is cut
Signal peptidase process
Once Sec61 opens laterally and the protein escapes –> the signal peptidase that recognizes the Amino Acid sequence near the signal sequence comes and cuts at that location
- Cutting off the signal sequence creates a new N terminal
In image – have the peptde in Sec61 and signal pepsidase binds and cleaves the signal sequence
What happens once cleave the signal sequnece
Once cleave the signal sequence –> the signal sequence stays in the ER membrene –> THEN another pepsidase (signal peptide petidase will come and cut so the signal sequence is removed from the ER membrane and break it down)
What happens after second peptide cut for a secreted protein
After cut the signal sequence = rest of the protein is translated –> protein will assemble and fold in the ER lumen
NOW the protein won’t be attached to the membrane because the signal sequence which was the transmembrane region was cut (NOW not anchored to the ER membrane)
After translation/folding the soluble protein is secreted out of cell (soluble protein will go in vesicle to the plasma membrane and then will be secreted out of the cell)
THIS IS MAKING SECRETED PROTEIN (protein will be secreted UNLESS there are more hydrophobic patches or other target information is present)
Ways to generate transmembrane/membrane-anchroed proteins:
- Additional hydrophobic patches on polypetide
- C-temrinal hydrophoic patch (with or without signal sequence)
- Additional other protein topoligies can be made if have a C terminal patch
- Exception – post translational translocation into ER membrane
What additional sequences does an integral membrane protein have
Intergral membrane protein has a stop transfer sequence (USES additional hydrophibic patch)
Integral membrane protein (tranemebrane protein) = has the signla seuqnece (start- stranfser seqnece) AND has another signla sequnece (hydrophobic patch) (2nd sequence = stop transfer sequence)
- Start transfer sequnce = same as orginal signal sequnece (hydrophibic Amino Acid residues)
- Have 2 signal seqneces (2 different hydrophobic amino acid patches on 1 polypetide)
What happens when a protein has 2 hydrophobic patches
When there is 2 hydrophobic patches –> proteins go to the ER –> Signal peptidase cuts the signal seqeunce (cut the start transfer sequence) –> rest of translation happens in the ER lumen –> get translation of the second hydrophic patch that coms into the traslocon and gets stuck in the ER membrane –> rest of translation continues in the cytoplasm = make a protein where there is N terminus in the ER lumen and teh C terminal tail is in the cytoplasm with a single transmebrane domain = makes a transmembrane protein
- The second hydrophobic patch does NOT have a cleave site = it gets stuck in teh ER membrane
What happens after translation of the second signal sequence
- SRP detects the signal sequence and stops tranlatiinon
- Ribsome goes back to ER and attches to the tranlcon –> signal sequence is inserted into teh tranlocon
- SRP detatches and translation continues
IF elbow first insertion - Ribsome stays on the tranlocon and forces more of teh protein into teh ER spac
What happens at the end of translation of transmembrane protein
Ribsome finishes tranlation –> protein is realsed from tranlacon
Protein finishes folding and is destined to be an integral membrane protein in teh cell or on teh cell surface
Overall what does a transmembrane protein has
Tranemebrane protein = has 2+ hydropbic patches
Image – the start transfer goes into Sec61 –> cleave signal sequence –> have tanslation until the second hydrophbic patch is translated –> patch gets stuck in lumen –> have loop of protein in lumen ; keep trasnlating in cytoplasm = end with C temrinal in the cytoplasm and N temrinas in teh ER lumen
Types of transmembrane proteins
Different types of transmembrane proteins can have different topology:
Type 1 – Single pass with the C terminus in the Cytoplasm and N terminus in the ER lumen (Ex. LDL recpetor)
- Have a cleaved signal sequence
Type 2 – N terminus is in the cytoplasm ; C terminus is in the ER lumen (Ex. Example – Asiaglycoprotein recpetor)
Type 3 – Have little N terminal in ER lumen and most of the protein and the C terminus is in the cytoplasm (Ex. Example – Cytochrome P450)
Type 4 – Have mulitple transmembrane domains (multipass transmembrane protein) (Ex. G protein coupled receptors)
When is topology of protein determined
Topology is determined during biogensis in the ER
Type = dictated by what is in the Amino Acid sequence
How do you know which side of protein is in the cytoplasm
General rule – net basic charge (Ex. Arg, His, Lys) is on the cytoplasmic side
- Arg, His, Lys = stays in the cytoplasm
WHEN the protein goes into Sec6 –> the basic net charge (positive charge) stays in the cytoplasm
The orientation is determined based on if have multiple basic residues (based on the Net charge)
Is the signal sequence always in the N terminus
The signal Sequence is NOT always at the N terminus
IF the signal sequence is not at the top of the N terminus then most of the N terminus is made in the cytoplasm by the ribosoms –> once the signal sequence is translated it is recognized by SRP and brought to the ER membrane and translocated by Sec61
- WHEN the protein goes into Sec6 –> the basic net charge (positive charge) stays in the cytoplasm
When is the N terminus vs. C terminus in the cytoplasms
IF the + charged amino acids are closer to the N terminus = N terminus will stay in the cytoplasm (+ AA/N terminus stays on Cytoplasm)
- If + is on the N terminus side of the signal = then the N terminus stays in the cytoplasm
IF have + charge after the signal sequence (closer to the C terminus) THEN the C terminus stays in the cytoplasm
- If the + charge is on the C terminus side of the Signal sequence then the C terminus stays in cytoplasm
What do multi-pass transmembrane proteins have
Multi-pass transmembrane proteins (poltopic membrane proteins) = will have multiple signal sequences or stop trasnfer sequences (ultiple start and stop sequences)
- Can have 7 transmembraen domains depending on how you want to make the protein
Hydropathy Plots
Hydropathy plots can help predict topology
Can look at hydropathy plots to see how many hydrophobic patches you have on the protein –> used to see how many transmembrane domains the protein has by looking at the hydrophobic residues on the peptides
- Now would just use alpha fold
Other approaches to determine membrane protein topology
Used to determine the orientation of proteins:
1. Accessibility of antibody epitopes
- Make AB against certain location on protein and see if it is accessible = Can see if that protesin is facing the extracellular space
2. Epitope tagging
3. Addition of N-linked glycosylation sites
- Can add glycoslylation sites and changes the size of the proteins = can see which way the protein is facing
NOW use alpha fold
Epitope taging for determining membrane topology
Use – can see which side the protein is facing
Can add epitope tag onto protein at a known locatiion and probe using an AB and see if it is accesible to the extracellular space or if you have to permeabilize cells
- Epitope tag - ex. His tag or small protein tag)
Having trasnmembarnes with a C-temrinal hydrophoic patch - WITH a signal Sequence
When have a having trasnmembarnes with a C-temrinal hydrophoic patch WITH signal sequence can use GPI anchor
With signal sequnece – GPI can be added to some type 1 transmembrane proteins in the ER to anchor proteins
GPI anchor Process
Once have the signal sequence go through Sec61 –> Cleave the signal sequence THEN make most of the protein the ER lumen and the last part of the protein will have a hydrophobic patch (red in image)
NOW the membrane domain is anchroed BUT becasue it can be cleave = you use GPI to anchor the protein to the membrane instead of the hydrophic AA to anchor the protein in the membrane
What are you using to anchor the protein in GPI
NOW instead of using Amino Acids to anchor the protein to the membrane a lipid anchors the protein to the membrane
- The protein is associated with the membrane
- When at the plasma membrane they are anchored but not soluble (NO secreted)
Shows GPI anchored protein using the secretion pathway
Making a transmembrane domain without a signal sequnece
Making a transmembrane domain without a signal sequnece – Tail anchored proteins are inserted into the ER thorugh the GET pathway
- Still makes a transmembrane protein
Example – SNARE proteins use this pathway to be inserted into the ER membrane
GET pathway
GET pathway –> All of the protein is made in the cytoplasm (all of the translation occurs on ribsoomes in teh cytoplasm) –> as translation is finished there is a hydrophibic patch on the protein –> patch is recognized by the pre-targting complex –> pre-targting complex brings the prtein to the GET3 ATPase protein –> GET3/protein binds to Get1/2 proteins on the ER membrane –> protein gets inserted into the ER membrane using ATP and the GET proteins –> NOW the protein is anchored to the ER membrane and can go through the secretory pathway
Making transmembrane/membrane anchored proteins post translational modifications
Done without the signal sequence (make transmebrane protein without the signal sequence)
Post translation translocation requries addtional machienry
There are other excpetiones (just know they exist)
Overall Review
Overall Review – have 2 types of cargos that go to the ER (secreted soluble proteins with no transmembrane domain and transmembrane domain proteins)
Both have a signal sequence BUT the transmebrane domain has signal sequences that is not cleaved or multple hydrophobic patches
Where are proteins folded and modified
Protein folding and post translational modifications happen in the ER
Uses:
1. Chaparones
2. Sugar Modifications
3. Disulfide bonds
Chaperones (overall)
Chaperones (Ex. Bip – member of Hsp70 chaparones)
Function - Chaperones helps the protein fold proteins in the ER lumen
Example - Bip function - Bip binds to hydrophobic patches on nascant proteins –> chaparones will folds the protein so the hydrophic residues are not exposed on the surface of the protein
- End – hydrophobic patches = hidden in the inside of the protein so that when teh protein is scereted into the aqeous envirnent the protein is still stable (because hydrophobic patches are buried Bip won’t bind anymore)
When does folding occur
Folding begins co-translatinoally and involoves chaparones
What proteins need sugar modifications
Have sugar modifications on proteins that are secreted
Glycosylation = adding sugar onto proteins
Glycosylation is important for secreted proteins
Where does glycosylation occur (what Amino acids)
Olgiosaccaride is added to aspargine residues found in a specifc conceceous sequence (Asn-X-Ser or Thr)
- Asparagin (Asn) gets glycosylated by proteins
Glycosylation happens by olgiosaccaried transferase (adds sugar to Asn)
Affect of sugar modifications
Sugar modification is large and hydrophilic –> influences protein folding
- Proper Glycosylation = important for correct protein folding for secreted proteins
Secondary affects of sugar modifications
Sugar modification is also important for:
1. Cell-Cell adhesion
2. Hydration of cell mebrane (hydration of cell surface bevcause sugar attracts water molecules)
3. AB specificities
4. Recognition by immune system
- Because bacteria proteins don’t have sugar = immune system knows this is a foreign protein and can target it
- Proteins humans make should have sugar modification = immune cells won’t recognize them as foreign
Glycosylation + protein folding
Glycosylation also acts a check point for protein folding
Disulfide bonds
Have disulfide bonds made on Cysteine residues on proteins because the ER is an oxidizing environment
- Disulfide bonds hapepn during translation in the ER because the ER is an oxidizinhg envirnmet
- Any cysteine can be disulfide bonded
Disulfide bond formation is an important folding step
What enzymes make disulfied bonds
Disulfide bonds = done by protein disulfide isomerase (PDI) (makes disulfide bonds correctley)
Affect of protein modifications
All modifcations affect how the protein folds –> protein folding is important for the function and secretion of proteins
Misfolded proteins
Misfodled proteins can be retrotrasnlocated and degraded
If protein folding does not happen correctley = have ERAD –> translocates unfoloded protein and then sends the protein to the cytoplasm for degredation
- Proper folding of proteins is important
When is everything happening
Eveyrthing is happening co-translationlly
When does clevage of the signal sequence occur
Durng translation the signla sequence is cut as teh sequence enters the lumen
Cleavge is co-tranlsation (as tranlation happens cleavage happens)
What sites will actially get glycosylated/disulfide bonded
NOTE – only glycosylate sites in the lumen + ONLY get dislfude bonds on sites inside ER
Practice
Set up – have the + charge (basic charge) on the N terminus + signal sequence with a cleavage site + 2 glycosylation sites + another hydrophic regions
After translation Answer – B
- Why B – cleave the instial signal sequence and then have the rest of tranlation in the lumen (stays in the ER) ; only glycosylate the sites that are in the lumen (other site is in the cytoplasm = not glycosylated)
During tranlsation answer – A
- Cleavge is co-tranlsation
- Answer is A –> When you cleave the signal seuqneve you have a new N temrinus and teh resy of the protein will continue in the cytoplasm
For after tranlsation answer – D
- No cleavage site on the protein –> signal sequence will act as a transmembrane domain
- Becuase the net charge is in teh N terminus = teh N terminus is expresed to the cytoplasm and the C terminus is in the ER lumen
For during translation – D
- Don’t have cleavge ; need the N terminus in teh cytoplasm and the trasnamenren in teh ER membrane
After tranlsation answer – B
- No hydrophobic patch once the signal is cleaved = soluble protein in ER lumen
During translation – A
- Have signal sequence cleaved co-translationally and the rest of the protein is made in the ER lumen
Experiment done for excersize #2
Question 2 – have a protein with 5 hydrophbic domians ; 1 cleavge site and have glycosulation sites and cystein resuduces that can make disulfied bonds when oxidized
Experiment (In vitro tranlation +/- microsomal memranes) – have microsome (purified ER membrane) –> microsome is added or not added (+/-) with all of the translation components (tRNA + ribosomes + intuation and elongation factors + AA) ; also adding radioactuve methionin (AA) into tube –> incubate and let the compoents make proteins in the tube –> treat as required –> denatures + reduces + probe for radiactiovity and do SDS page –> look at protein size (only the radiolabled protein is detected)
Microsome
Microsome = purifed ER with ribosomes
Interpretting Gel
Lanes:
1. Have no detergent, no protease, No Glycosylase inhibtor , no Mebrane –> Just mRNA and ribsome and tralation thing in tube
2. Have No detergnets, No poreteases, No glycosylase inhibitor, WITH membranes
- Added mebrane and now get largere protein (bigger protein = adding something to the protein)
3. Have No detergent, No proteases, WITH glycosylase inhibitor (NO glycosylation), and WITH membranes
- Inhibit glycosylation and have mebrane and end up with protein that si smaller than the first lane
- Tells you that glycosylation is causig ashift in teh lane and there is an extra down shift in the thrid lane because the signal is getting cleaved
4. Have No detergent, WITH proteases, No glycosylase inhibitor, WITH membranes
- Adding proteases = cuts protein (get 3 peptides on diferent sizes)
5. Have No detergent, With preteases, With Glycosylase inhibitor, With membranes
Use of adding detergent in last lane
Acts as control –> shows protease works (because have no protein which means when degraded the membrane the protease was able to derade all of the protein)
Lane 5 vs. 6
Lane 5 vs. 6 = added a glycosylase inhibtor (have 1 glcysykatin site that is able o be glycosylase in lane 5 but not 6 because the land 5 band s bigger)
Adding teh sugar to the second of the 3 bands
What is the topology of the protein?
Answer – A
KNOW that theer are 5 transmembrane domains BUT 1 gets cleaved = theer are 4 transmembrane regions = ignore C and D
N terminais is cleaved (N terminus is in teh ER once cleavages occurs)
- Needs to be A because of the orientation
- Issue in B = have too many glcosylation sites inside lumen (diff between lanes = 1 glcysylation = need to have1 glycosylation site in teh ER whcih A has)
ISSUE - try and figure out what it woudl look liek with N terminus on teh outside
Answer – Site A
Glycosylation only adds 2.5 kDa = need only 1 glcyosylation site = only site A is in the ER
ON PAST SLDIE – if B was rght = have 2 glycylatoon sites vs A only has 1 glycosylation site in ER
Answer – E
Any resudes in ER can form disufide bonds (any of these three optuons could work because they are all in the lumen – 1,2,4 are all in the lumen)
How do you now protein structure and localization
Based on protein Amino Acid sequence –> can predict the protein strcuture and localization
Process in video (make sure you know BUT eveyrthing here should be in past cards)
Start - mRNA in cytoplasm
1. Free rivbosome will bind to the mRNA and begin tranlation (
2. First signal sequence exits the ribsoome
3. Singal recognition particle (SRP) bind to the signla sequnce –> binding of SRP pauses translation
4. SRP ribssome complex (SRP + ribosome + mRNA) will go to the ER membrane
5. Once at the ER membrane –> SRP binds to SRP receptor –> protein sequence is located into the tranlocon
6. After the protein is inserted into the translocon teh SRP will detatch
7. Once SRP leaves teh ribsoome starts translation again
8. Ribsome will detcah from tranlocon and will continue tranlating in the cytoplasm ; AT the same time the glycosylation domain is recginzed by ER prpteins = add sugar to the proteiin
- During tranlsation the tranlocon will open a pore (in like side of the tranlocn) to eject the hydrophobic domain of the proetin into teh ER membrane) frees the translocon
- Protein will move laterally in teh ER membrane (the tranmembrane domain stays in the ER membrane but the proptein move over to the side)
9. Second signal sequence is created
10. SRP detects the signal sequence and stops tranlatiinon
11. Ribsome goes back to ER and attches to the tranlcon –> signal sequence is inserted into teh tranlocon
12. SRP detatches and translation continues
13. Ribsome stays on the tranlocon and forces more of teh protein into teh ER space (because of elbow first insertion )
14. 3rd hydrophib domain enters tranlocon with teh positive AA towards teh cytopalsm = cuased the ribsome to detatch from the tranlsocon (have head first isnertiion)
- Head first because the postive amino acids aready face the cytopalsm = want to keep it that way
15. Ribsome finishes tranlation –> protein is realsed from tranlacon
16. Protein finishes folding and is destined to be an integral membrane protein in teh cell or on teh cell surface
Decision that gets made when protein is inserted into the Sec61
Insert the protein head first (N terminus down – N terminus goes into Er lumen)
OR insert elbow first (N termus would be in the cytosol)
Decsion = based on posotivley charged Amino Acids
- Side of signal sequence with more + charged amino acids is kept in teh cytoplasm
I think head first = N termius goes straght down into teh ER lumen IF there are positive AA closer to the C terminus ; the protein is insetred elbrow first (flips teh proetin so now the C terminus is in teh ER lumen if there are + AA near the N terminuis)
Translation after head first insertion
Because protein was inserted head first into the tranlocon the ribsome must contyinue creating the protein in the cytoplasm to align with the head first insertion
Translation after elbow first insertion
Ribsome stays on the tranlocon and forces more of teh protein into teh ER space (because of elbow first insertion)
