Co-translational localization to the rough ER
Research Question • IL-1, a secreted lymphokine that indudes fever upon infection, lacks a signal sequence from its primary amino acid sequence. Devise an approach that can be used to ascertain the mechanism of IL-1 secretion.
Overview
• Eukaryotic cells are structurally and functionally compartmentalized. • This provides a favorable chemical environment for specialized reactions to take place in a large cell. • Specialization is provided by proteins that are sorted (localized) to these compartments.
Signal sequences for secretion
Organelles are specific for eukaryotes
Organelles have specific fxns provided by the proteins moved specifically to those organelles
Translocation: moving a protein across a membrane into an organelle
Protein sorting mechanisms
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Another word that people use for this subject is protein sorting: this means that all the proteins are made in free rib or rough er
Cell has to sort and send out to right location
Transport for protein
Cytosol to nucleus: gated transported: nuclear pore made of a bunch of proteins and regulated by GTP hydrolysis; the whole proteins was already synthesized by free ribosomes and moved through nuclear membrane through the gate

Proteins always first in cytosol.
Proteins from cytosol to ER then into mitochondria or chloroplast or peroxisomes etc depending on where they are going
Through a transmembrane transport using and signal sequence that targets them into the right organelle
Once in rough ER then to Golgi and cell surface via vesicle
Proteins that enter mitochondria directly not go to the rough ER
Lipids made in smooth ER but here they are talking about the rough ER

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All of the mRNA are going to begin translation in the cytoplasm
Review translation on own
Translation begins on free ribosomes in cytoplasm and depending on where they are going to be targeting; translation in cytosol or stalled until they get to rough ER membrane
Cytoplasmic proteins are translated in cytosol (they are not getting more modification like DS bonds)
Proteins that go into the rough ER are the ones that are going to be in the membrane or secreted and then Golgi( vesicles

Removed after sorting
Not removed after sorting
Proteins are sorted to organelles according to signal sequences found in their amino acid sequence.
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Proteins are sorted into organelles by having a signal sequence
A signal sequence, if it is going to be moved into a organelle is going to be in amino terminus
Most proteins the amino end will be used for signal peptide (signals where to be translocated)
The first case A the signal sequence is removed when the protein is translocated across the membrane
For some sequence its not a signal sequence in amino terminus
The signal sequence that is used to translocate the protein has to come together when the protein is folded for it to be translocated
Signal patch is evident when protein is folded and not removed until it gets to destination
The patch is a 3D structure
First case the sequence is at the amino terminus and not coded and it targets protein into an organelle
In 2nd case the patch is not in a defined location in the amino terminus; it is only evident when the protein is folded (in 3D structure) and that is what is recognized when it gets to target

Signal sequence is cut out but patch is not; you wont see a difference between two lanes if you run it on a gel

Examples of signal sequences
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Don’t have to memorize Techniques used to study protein sorting • Transfection of recombinant proteins with localization signals • Protein translocation, cell-free assays • Genetic approach • Microscopy • Ultracentrifugation of vesicles Determine how proteins are sorted in cells One of the techniques is transfection: using GFP or LacZ as a marker and cloning a signal sequence upstream( transfect into eukaryotes the protein will translocate into the right place (in vivo) Cell free assay: isolate rough ER, Golgi and vesicles to remove everything in cell and follow movement Genetic approach: sensitive mutants?
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In this case you have a construct of plasmid that has either a cytosolic protein that’s not found in the rough ER and a signal seq for that organelle
The construct has DNA sequence for the signal sequences for that organelles but the protein is cytosolic
Transfect into a cell and expect the cytosolic protein will be in the rough ER
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Cell free assay:
In one case you can label the protein radioactively
You have protein with a signal sequence and you can mix vesicles from the ER and mix the proteins with the signal sequence that is labeled and you will expect the protein to translocate to vesicle and you expect the signal sequence to be removed during translocation( check by running a gel; you determine if the signal peptide is removed (if removed it is smaller)
You only label the protein you want
You can use an antibody against the protein if you don’t want to radioactively label
Where in the process would you use the antibody? You use a western blot after you run in a gel
There are markers you can use to determine whether the fraction came from the Golgi, rough ER or cytoplasm—this is one way to look at the antibody
What is another way without using the gel and looking at the antibody in the cell
Use transmission electron microscopy with gold labeled antibody to look at location of that protein (FACS will only tell you if the protein is there and not the location; in situ only tells you about RNA and not the protein)
Bottom part: they also check whether the protein was translocated into a vesicle; if you add protease the protein will be broken up and you don’t see anything in SDS page; if the protein did move into a vesicle then the protease will not digest—then you break up the vesicle and run on gel to get proteins
If you add detergent you expect the protein to be degraded if you incubate the protein with trypsin then you cut the whole protein; if it is in the vesicle then you don’t cut it
Use detergents to make sure that protein was inside the vesicle and not misfolded or aggregated

What info do you get in this slide? 1. Does the protein translocate? 2. Does it require a signal sequence?

You incubate the vesicles with the protein how do you know it has translocated? You add proteolytic enzyme and compare to “control” protein of original size in lane 1: in lane 2 you have labeled protein + vesicles + proteolytic enzymes but it is a little below control in lane 3 it is the same as 2 plus detergent and what you get is a spot towards the bottom
What do lane 2 and lane 3 tell you about the cell? You disrupted the vesicles with detergent then the enzyme will get in and cut the inner part of the transmembrane protein; but you have to remember that detergents surround the hydrophobic so what you see in lane 3 is the part of the protein that is embedded in the membane
What does lane 1 and 2 tell you? That some of the protein is outside (it is transmembrane) and that outside part is being cut
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Genetic method:
Temperature sensitive mutants
This is specifically used for protein translocation
In this case, you have yeast cells and a protein
Protein that synthesizes histadine is in the cytoplasm, will not go into rough ER
You can make a mutant that has that enzyme coupled to a signal peptide that would translocate it into the rough ER(transfect into yeast cells (create mutant) then the enzyme will move into rough ER and the cell will not be able to use histadinol to make histadine so if you remove the histadine from the media then the cell will die
Way to test proteins in translocation and translocaton in general

Creating mutant involving the enzyme in histadine and see if translocate
Induce and screen for mutants where the enzyme was not moved into ER at a higher temp
The enzyme that was engineered with a signal peptide will translocate under low temp but not high temp
Enzyme has signal peptide if you grow that cell at lower temp then it goes into rough ER and no his in media the cell dies
At higher temp the protein will stay in the cytoplasm (looking at the proteins involved in translocation—protein is there but misfolded) and the cell will live bc what is misfolded is the translocation proteins but not the enzyme itself
The mutant can make the protein and fold at low temp and you get fxn; if you increase the temp then the protein will be misfolded and you lose the function of the protein
Why is this system good for looking at phenotypes? You still have the cell and you can look at the function

Temp sensitive mutants
Looking at proteins involved in translocating proteins into the rough ER
First you introduce to enzyme that is synthesizing histadine which is always in the cytoplasm; if you add the signal peptide and it goes to rough ER then the cell will die bc it doesnt have his
In experiment they induce mutations and select mutants which at lower temp cannot synthesize histadine (they dies bc the proteins in translocation work the enzyme goes into rough ER)
At higher temp the transport proteins will misfold and the enzyme is not transported/translocated into the rough ER
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Using fluorescence to look what sequence are in nuclear localization
What would you use GFP
If you clone and nuclear localization signal upstream of GFP and it will be translated and it will be moved into nucleus
In B if you mutated a nucleotide in the AA and the protein stays in cytoplasm and fails to go into nucleus

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Ultracentrifugaton to sep smooth ER and rough ER; if you are doing functional assays you have to use vesicles from rough ER
The rough ER has ribosomes and more dense that smooth ER
This is way to get rough ER vesicles

Protein translocation • Two types: • Co-translationally • Into the rough ER (not 100%) • Post-translationally • Into the mitochondria, chloroplasts, nucleus, peroxisomes Moving into the rough ER is mostly cotranslational (not 100% but most)
In the other organelles like mitochondria the proteins are moved post translational

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Translation always occur in free ribosomes in cytoplasm (at first)
The peptide sequence is hydrophobic in amino terminus so therefore it has to be protected from aqueous cytoplasm
SRP (complex of RNA and protein) bind to signal sequence and stops translation until the whole thing is brought into Sec61 which is a complex that forms a pore in the rough ER
Once the complex (signal and ribosomes) into rough ER, docks and continues translation and signal sequence is cut by transmembrane signal transpeptidase
The protein get synthesis and is folded in the rough ER
Folding: proteins cannot fold on their own( they use a folding box (also called folding cage)---there will be a handout of this
This folding box is made of proteins belonging to the HSP family of proteins and form a large structure that uses ATP to fold proteins
In vitro you can only fold small proteins and in vivo most proteins are folded using this box
Chaperones take the protein to the cage and the inside of the box is hydrophobic and the hydrophobic parts of the protein interacts and then ATP is used to change conformation of box from hydrophobic to polar and now the hydrophobic has to interact with themselves; many cycles

Mechanism of Action
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SRP (6 proteins and a small RNA) bind to signal peptide)( translation is stalled (ribosome binds to the rough ER( translation continues( protein is translocated to the rough ER lumen.
SRP: small RNA with 6 proteins; bind and translation is stalled and they bring the whole thing to rough ER
This is a structure of the SRP complex; it is a complex bc you have an RNA
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RNA and 6 proteins that form the complex
There is a sequence that binds
Other side stalls translation
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The SRP binds to the signal peptide on the new protein (which is translated by ribosome) and the SRP twist and bind to large ribosomal subunit to stop translation.

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Its not like the complex floats and find ribosomes; there are microtubules involved in moving it
The SRP recognized hydrophobic signal peptide
The complex binds to the SRP receptor protein and translocatar binds
The translocater on rough ER membrane binds to signal peptide and allow release of SRP
Once SRP leaves then the ribosome is free to translate
Go through pore into rough ER lumen
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The translocater is sec61
Complex made of multiple
Alpha helix in the middle is a gate (gated transporter) so when the translocater is not in use the plug blocks access to lumen of rough ER
When the SRP is brought onto rough ER membrane and binds to SRP receptor the signal peptide is going to open the gate and it is going to allow that protein to enter through the pore into lumen of rough ER
Only open when signal peptide is interacting
The channel in the large ribosome where you get elongation is directly oriented into the pore so as long as SRP left the complex then the ribosome can elongate the protein through the pore
The parts of the translational machinery interact with transporter
The whole process is conserved throughout evolution in eukaryotes and bacteria
Where would you only see the proteins translocated in plasma membrane in bacteria (no organelles) you have a protein with a signal sequence that is recognized by a complex and it moves the sequence to plasma membrane where it is going to be secreted by cell or go into membrane; in eukaryotes the ATP is used to fold protein that goes into translocater; in bacteria the ATP is used to push the proteins through membrane

Once docked then the signal peptide leaves the complex to interact with translocater

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Signal sequences of amino terminus is going to be translocated and but by signal peptidase and goes into rough ER (secreted proteins)
Paper on website about folding in ER not responsible for; other paper responsible for
Translocation across rough ER membrane
There are diff ways the protein can translocate into rough ER depending on where the signal sequence is located
If it is at the amino terminus then it is what you see here; it is the first sequence that is going to be translated; SRP binds and stalls; docks on membrane and then the signal peptide opens the gate on the translocater and peptidase cuts signal and it translocates into rough ER—this works for secreted, lysosomal, soluble proteins

Co-translational transport of membrane proteins • Two signal sequences • A start transfer sequence (cleaved off by signal peptidase) • A stop transfer sequence (transmembrane region) • Internal start transfer sequence • Not cleaved off • Two signal sequences • Not cleaved off
Signal sequences of amino terminus is going to be translocated and but by signal peptidase and goes into rough ER (secreted proteins)
Paper on website about folding in ER not responsible for; other paper responsible for
Translocation across rough ER membrane
There are diff ways the protein can translocate into rough ER depending on where the signal sequence is located
If it is at the amino terminus then it is what you see here; it is the first sequence that is going to be translated; SRP binds and stalls; docks on membrane and then the signal peptide opens the gate on the translocater and peptidase cuts signal and it translocates into rough ER—this works for secreted, lysosomal, soluble proteins

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Two signal sequences: start transfer in amino and stop transfer in the middle
Start signal cut out
Stop trans not cut out; it is going to ensure the protein is embedded on the membrane; The ribosome will keep translating but that portion is not going into rough ER once you hit the stop transfer signal
Which part is going to be outside? The amino terminus going to be glycosylated in the rough ER and Golgi in the amino area so the amino end is going to end up in the cytosol

The proteins that recognize the stop sequence the ribosome is going to dissociate

The outside residues are positive

Transmembrane proteins; there are two sequences—start transfer at amino and SRP binds and stalls translocation and also hydrophobic again
The protein is translocated into rough ER into hydrophobic portion is embedded and the remaining translated outside
N terminus inside rough ER and C terminus outside in cytoplasm; what is inside is glycosylated

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You see amino terminus facing the cytosol and C terminus facing ER
When you have a stop trans sequence the amino acid that faces the sequence tells you something about the orientation
If you have negative around the stop then it is going to face ER
If you have positive around the strop transfer it is going to face cytosol
If the residues before the stop sequence must be negative and the other side must be positive
Protein only has one signal sequence in the middle then the charge has to do with the orientation of the protein

One single sequence in the middle: orientation depends on the context
In this case the part of the protein that goes into rough ER has negative charges AA closest to signal sequence other side are positively charged AA; this only works for signal sequence in the middle (only this type)
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[pic] here you have a start and stop signal in one sequence
If the sequence in the middle of the protein it is not cut out; the only way the start transfer is eliminated is if it is at the amino
Many transmembrane domains here

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Proteins that enter the rough ER are glycosylated.

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Glycosoylation starts in rough ER and contain in Golgi
These proteins are involved in cell-cell recognition; neighboring cells in same tissue have same glycosylation

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If protein is transmembrane then gly only in rough ER
Two types of glycosylation:
N-linked glycosylation: amino acid that is glycosylated is asparagine and (asp—x---ser or asp ----x---asp) this is actually a seq that is going to be glyc; its not like if you have asp it will automatically glycosylate all of them; has to be in certain sequence
O-linked glycosylation: lysine, hydroxy-lysine (modified AA)
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If protein is transmembrane then gly only in rough ER
Two types of glycosylation:
N-linked glycosylation: amino acid that is glycosylated is asparagine and (asp—x---ser or asp ----x---asp) this is actually a seq that is going to be glyc; its not like if you have asp it will automatically glycosylate all of them; has to be in certain sequence
O-linked glycosylation: lysine, hydroxy-lysine (modified AA)