MCB 150 Week 6 topic 1: fates of translated proteins

describe the process and purpose of N-linked glycosylation.

1. A lipid called dolichol is made, and it is in the membrane of the ER. 2. A 14mer oligosaccharide tree is built one monomer at a time onto a molecule of dolichol. 3. As the protein is being made by a membrane bound ribosome and it is coming through the translocon, there will be a particular stretch of three amino acids, one of them being an asparagine. 4. The 14mer is transferred from dolichol onto the asparagine. Eventually the 14mer is trimmed to an 11mer. This signifies that the protein if fully translated, folded and ready to move on. 5. The 11mer can be further modified; these modifications can act as a signal for where the protein needs to go (for example a phosphate group can be added to the 6th mannose in the golgi, to form mannose-6-phosphate group, which now says "take me to the lysosome").

Identify the three things that SRP binds to, and provide a reason why each is relevant.

1. The signal peptide: recognizes the signal that says "I need to be translated at the membrane of the RER." 2. The ribosome: Stops the ribosomes from translating the rest of the protein while it is still in the cytosol. 3. SRP receptor: This is the "dock" where the ribosome is now located at the RER. The binding of the SRP to the receptor (and another step that requires GTP) allows the SRP to release the ribosome and the signal peptide, so that it can be used again. This release additionally allows translation to begin again, this time feeding the protein into the translocon (which was opened when the ribosome bound to the translocon).

The Pulse-Chase experiment (1960): how its done, pulse levels, how is S35 incorporated in?

1. give a culture of actively growing cells a large amount (a pulse) of a radio-labeled amino acid a pulse is a level of radioactivity that's detectible but doesn't interfere with cell activity: radioactive sulfur is good to use (S35): found in methionine and cystine during translation some of the radioactive S35 is incorporated into the growing protein chain where there is a methionine 2. now we must chase away the radioactivity to STOP incorporating the S35. Now we're going to flood our culture of growing cells with an excess of cold, which means not labeled version of that amino acid.

2 ways to make translocon close

1. presentation of stop-transfer sequence 2. lifting off the free ribosomal unit

Order the following events, from earliest in the process to latest in the process, of a protein with an amino-terminal signal peptide being translated and folded in the ER lumen. The signal peptide gets removed by signal peptidase SRP docks on the SRP receptor and the ribosome is lined up over the translocon The protein is bound by BiP and is correctly folded The translocon opens and the ribosome resumes translation The SRP and ribosome are moved to the RER Signal peptide emerges from ribosome SRP binds to signal peptide and translation is halted

1. signal peptide emerges form a ribosome 2. SRP binds to signal peptide and translation is halted 3. The SRP and ribosome are moved to the RER 4. SRP docks on the SRP receptor and the ribosome is lined up over the translocon 5. The translocon opens and the ribosome resumes translation 6. The signal peptide gets removed by signal peptidase 7. the protein is bound by BiP and correctly folded

3 primary responsibilities of RER

1. synthesis of secreted proteins 2. synthesis of transmembrane proteins 3. modification of proteins that make their way through the secretory pathway

If all proteins are translated in the nucleus, why are some proteins also "translated" on ribosomes on the surface of the endoplasmic reticulum?

A ribosomes in the cytoplasm translates mRNA but the protein, as its made, is threaded into the interior of the endoplasmic reticulum, also known as the lumen. The ribosome never enters the lumen but instead attached to the membrane

Where does lipid biosynthesis occur?

All of those precursor molecules are going to be assembled and integrated into a phospholipid by an enzyme that's bound to the cytosolic leaflet of the smooth ER membrane. In other words, this membrane-lipid synthesis happens at the smooth ER but not inside the smooth ER. It actually happens on the cytosolic side of that smooth ER membrane.

most of the proteins that are ER resident (live and work in the ER) are responsible for protein processing and folding. what are some examples? (2)

BIP: chaperone assisted folding Signal peptidase

When is the transmembrane protein's N terminus in the lumen and C terminus in the cytoplasm? AMINO TERMINAL SIGNAL PEPTIDE

C-terminus in the cytoplasm because the ribosome finished translation while the protein had already been ejected from that translocon. N-terminus is in the lumen because the signal is ON the amino terminus signal sequence is amino terminal folded in with N terminus facing cytoplasm and C terminus facing lumen. we ARE cutting off internal signal sequence

How does the liver use the smooth ER for detox?

Dfetoxifying enzymes that are found in the lumen of the smooth ER can take what would potentially be harmful lipid-soluble molecules; traditionally the molecule that's used as the classic example would be barbiturates like phenobarbital. These molecules are lipid-soluble and interfere with metabolism, so those drugs get moved to the liver where enzymes in the lumen of the smooth ER convert those lipid-soluble molecules into water-soluble molecules that can be then removed from the body in the urine.

True or False... adding radioactive S35 degrades all other parts of the protein chain that does not include methionine

False. S35 is weak enough to not interfere with the protein but strong enough to be detected. Therefore, the S35 simply marks the methionines while the rest of the protein chain remains the same

The Pulse- Chase experiment (1960): background and why we do it

George Palade developed way to trace the fate of protein using technique he called Pulse-Chase experiment Got him Nobel Prize in 1974 why we do it: protein synthesis is going on all the time in the cell so if experimenter wants to focus in on one specific protein it's hard. SO you mark set of proteins and watch only those

The SER plays roles in... (2 things)

Lipid bilayer synthesis Detoxification

Which one of the following statements about protein modification in the ER is incorrect? Lipid-linked proteins are attached to the membrane via hydrophobic interactions between nonpolar amino acid residues. In N-linked glycosylation, a 14-mer carbohydrate tree is covalently added to an asparagine found in the polypeptide chain. Lipid-linked proteins synthesized in the ER are exposed on the cell surface. BiP molecules as well as enzymes that catalyze disulfide bond formation perform their functions exclusively in the lumen of the ER. Lipid-linked proteins made in the ER will never face the cytoplasm.

Lipid-linked proteins are attached to the membrane via hydrophobic interactions between nonpolar amino acid residues. they're covalent interactions

Why do we alternate signal peptides and stop-transfer sequences?

Logically you can't open what's already open. You can't close what's already closed, so you would never follow a stop-transfer sequence with another stop-transfer sequence. That doesn't make any sense. You would never follow a signal peptide with another signal peptide.

Does the addition of "cold" amino acids change the rate of protein production in a cell?

No, the cell makes the same amount of protein we just aren't going to see them.

Would you expect the amount of smooth ER in testicular tissue to remain relatively constant over the life of a male human? Why or why not?

No. Cholesterol is lipid precursor for steroid hormones, such as testosterone. Lipids are made in the SER. As more testosterone is produced, more SER would be needed.

when a transmembrane structure is formed, does the ribosome know or care if the translocon is opened? what happens to the ribosome?

No. It continues to translation until it's told to stop with stop codon.

Explain the experimental reason why Palade did not include mitochondria in what is called the Secretory Pathway.

Palade used pancreatic cells, which secrete a lot of proteins outside of the cell. The bulk amount of proteins made in these cells follow the secretory pathway and he therefore saw the proteins go from RER→Golgi→Vesicles→extracellular space. The proteins that get secreted are all made by membrane bound ribosomes. Mitochondrial proteins, on the other hand, are made by cytosolic free ribosomes and then transported to the mitochondria, and are not part of the secretory pathway.

What did the Pulse-Chase experiment show about how protein production is organized?

Protein production takes place in specific spots in the cytoplasm and it's localized supported the hypothesis that secreted proteins are translated in a central location and moved as a group through some sort of a pathway until they are all exported out of the cell.

The RER plays roles in...

Protein secretion Synthesis of membrane proteins Protein processing

Which one of the following statements comparing the targeting of a mitochondrial matrix protein to the targeting of a protein to the ER is correct? The same signal can be used to direct a protein to either compartment, as long as the appropriate receptor recognizes the signal. Proteins destined for either compartment are likely to retain their amino terminal targeting signals. Proteins destined for either compartment are inserted into their respective compartments in an unfolded state. Proteins translocated into either compartment have an equal chance of ultimately being localized to the outside of the cell. Proteins destined for either compartment are allowed to fold in the cytoplasm and then are unfolded prior to insertion into their respective compartments.

Proteins destined for either compartment are inserted into their respective compartments in an unfolded state.

How do cells direct proteins to the proper location?

Proteins that stay and function in the cytoplasm have no additional "targeting" signals Proteins destined for the nucleus have a nuclear localization signal Proteins destined for a mitochondrion have a mitochondrial presequence

What do the smooth and rough ER look like physically?

RER:flattened sacks of membranes with the studded ribosomes binding to them SER: system of connected tunnels

What is the path of secretory proteins determined by the Pulse-Chase experiment? A.K.A. secretory pathway

Rough Endoplasmic Reticulum --> Golgi Apparatus --> vesicles --> exterior

Which structures play a direct role in the production and localization of a secreted protein?

SRP, signal peptide, BiP

SRP

Signal Recognition Particle: 1.recognizes an ER directed signal peptide and binds to it on the emerging protein 2. can also bind to the ribosome itself so it also grabs the ribosome, stopping it from continuing the elongation phase. like hitting the "pause" button 3. makes its way to nearest RER membrane and binds to the SRP receptor. "dock" for SRP. sets ribosomes on the translocon (similar to TIM or TOM) only wide enough for unfolded protein. also makes the SRP molecule let go of the receptor, the ribosome, and newly emerging protein. 4. SRP leaving causes translocon to open and translation resumes but emerging protein goes down through translocon into lumen of ER 5. consensus sequence recognized by signal peptidase that recognizes the junction between the peptide that was acting as your signal and the rest of the protein and chops at that junction. you're sort of feeding in the signal peptide, and it's getting stuck to the inside of the translocon as it goes in. So then the C-terminal end of the peptide is going to be in the lumen, where signal peptidase can chop it.

What two membranes is the RER membrane continuous with?

The Smooth ER and the outer membrane of nuclear envelope with one continuous internal space between each ER lumen of ER is continuous with space between outer and inner membrane of nucleus (perinuclear space)

What ultimately decides the final location of a protein?

The site of translation. Some proteins translated fully out in the cytoplasm

What targets translation to the ER? instead of just allowing it to stay in the cytoplasm?

Theoretically, it could be either the mRNA, ribosome, or protein that's presenting some sort of signal that says, "This shouldn't continue out in the cytoplasm. We need to stop what we're doing right now, move over to the ER, and then we can resume at that point." ribosome: nothing different between ribosome translating in RER versus translating in cytoplasm so thats not it mRNA: NO because all translation initiates on cytosolic ribosomes protein: different after you get started versus before so has the sequence of amino acids that presents 3D shape acting as signal to finish translation on RER

A transmembrane protein at the plasma membrane crosses the membrane six times and has both its N- and C-termini in the cytoplasm. Identify the targeting signals, whether present at the plasma membrane or not, that directed the cell to orient the protein in this manner.

There was no amino terminal signal sequence. The first sequence presented was an internal signal sequence. In the plasma membrane there are three internal signal sequences and three stop transfer sequences.

N-linked glycosylation

Virtually every protein that goes through the ER goes through this specific process and in most proteins happens in a variety of different locations the addition of an oligosaccharide tree, or group of sugar molecules, to a specific asparagine residue, or amino acid, within the primary sequence of the protein that is making its way through the translocon 1. protein presents a signal peptide and gets directed to the ER. As it is being inserted through the translocon, a special sequence of amino acids is presented as a signal. This is different than a signal peptide; it's different than a stop-transfer sequence, and it's only three amino acids, one of which is an asparagine. Now that asparagine has a nitrogen in it, which is why this is called N-linked glycosylation 2. build fourteenmer (oligosaccharide) tree on dolichol platform 3. enzyme recognizes asparagine residue and prepared fourteenmer and cuts fourteenmer off dolichol platform and link it covalently to the three amino acid sequence 4. trim fourteenmer down to elevenmer by trimming the glucose off. signal that protein is now ready to go to the next location once its done being translated

can signal peptidase tell the difference between internal signal peptide and amino terminal signal peptide? If so which one does it cut?

Yes. the signal peptidase does not touch the internal signal peptidase but cuts the amino terminal signal peptide

How do we get a protein into transmembrane structure?

additional signal. stretches of amino that tells translocon to do something different. called stop-transfer sequence

GPI (Glycosylphosphatidylinositol) anchoring

also occurs in the RER: makes lipid linked protein can happen to a protein that's already been N-linked. only small subset of proteins get GPI anchors 1. prepare protein and catch it in the membrane so it doesn't get away from us 2. prepare the GPI anchor. PI stands for phosphatidylinositol, which is a major membrane phospholipid. that phosphatidylinositol molecule is now going to have some sugar groups associated with it, covalently. when you glycosylated a phosphatidylinositol you get a molecule called glycosylphosphatidylinositol. And that's a mouthful, so we just abbreviate it GPI 3. bring together the GPI anchor and the protein. there is an enzyme that recognizes a prepared GPI anchor, recognizes a protein with a specific C-terminal tail that has been held into the membrane. It's going to cut the C-terminal tail off of protein and hold on to the new C-terminus and link it covalently to an ethonolanine groups in the GPI anchor.

Why is an integral membrane protein even though no part of it is embedded in the membrane?

because we can't just float away the protein part. If we were to remove this protein from the membrane, because its connections all the way down to the phosphatidylinositol are all covalent, we'd have to pluck out the phosphatidylinositol, and that would disturb the integrity of the membrane

BiP

chaperones that bind to proteins and fold them once they are in the lumen of the ER

The Signal Hypothesis

created by Bernhard Dobberstein and Gunter Blobel proposed that of the 3 molecules innvolved in translation (mRNA, ribosome, and protein), the protein is the one to present a signal in the form of a 3D sequence of amino acids that tells the cell to finish translation on the RER

what does "endoplasmic reticulum" mean? use roots of the word

endo= within (the cyoplasm) reticulum= a network a network of membrane bound tubes and sacks

flipase

flips half of the phospholipds from one leaflet to the other to balance out the number in each leaflet.

What do we ned for a new phospholipid?

glycerol a couple of fatty acid tails a phosphate group other chemical group to be covalently linked to the other side of that phosphate

What does it mean for an amino acid to be "hot" and "cold"?

hot: it's radioactive. usually with S35 and therefore detectable. labeled version of amino acid cold: not labeled version of amino acid

Bernhard Dobberstein and Günter Blobel

hypothesized that there is a specific amino acid sequence that targets proteins to the ER: referred to as the Signal Hypothesis

Where are all proteins translated (at least at the beginning)?

in the cytoplasm

How is a multi-pass transmembrane protein formed?

initiates in same way as single-pass transmembrane proteins depending on where the signal peptide is. if you don't present an additional signal you will continue the status quo...so you must trap the C terminus or N terminus in the membrane by closing the translocon before translation is over by using a stop-transfer sequence

Proteins fully translated on cytosolic ribosomes (in the cytoplasm) that aren't attached to membranes go to...

just stay in the nucleus (no additional signal) the nucleus, mitochondria chloroplasts peroxisomes

Dolichol

lipid that gets phosphorylated a couple of times that gets sugar molecules attached to it putting together oligosacchraride tree that has 14 monosaccharides (fourteenmer)

what is the one chemical environment in the cell suitable for disulfide linkages?

lumen of the RER. any protein that has disulfide linkages in it had the disulfide linkages formed in the RER

stop transfer sequence: what type of amino acids usually and what does it do?

mostly hydrophobic amino acids because crosses interior of a lipid bilayer gives a set of instructions to the translocon to eject the protein out of the translocon into the membrane

How do you make a translocon open?

signal peptide

multi-pass transmembrane protein

span the membrane several different times and has either its C or N terminus in the lumen and the other in the cytoplasm N and C terminus could both be in either the cytosol or lumen

single-pass transmembrane protein

spans the membrane and has either its C or N terminus in the lumen and the other in the cytoplasm

Proteins translated on membrane bound ribosomes attached to the endoplasmic reticulum go to....

stays in the Endoplasmic reticulum (no additional signal) plasma membrane secretory vesicles: on way outside cell endosome, lysosome golgi appartatus

the more smooth ER you have.. the more _____ you have to accommodate the enzymes that convert cholesterol into those steroid hormones.

surface area

How do we fix the imbalance between the two leaflets after new phospholipids are added to the membrane?

take half of the phospholipids we just made and move them to the other leaflet. this is energy dependent because energetically unfavorable: need enzyme called flipases

lumen

the interior compartment. general term. the ER doesn't have a specific name for the inside so just called the lumen

only reason you'd make a GPI anchored protein?

to present it to the outside world. so it'll go through secretory membrane through plasma membrane to exposed to the outside world

When is the transmemberan protein's N terminus in the cytoplasm and C terminus in the lumen INTERNAL SIGNAL PEPTIDE

we must still have a signal to tell us to move to RER but the signal is NEAR the amino terminus signal sequence becomes internal in the membrane folded in with N terminus facing cytoplasm and C terminus facing lumen. but NOT cutting off internal signal sequence N terminus left out in the cytoplasm and ribosome dissociates making the translocon closes and squeezes out anything in it: which in this case is the internal signal peptidase

When does Lipid biosynthesis occur/for what reason?

when its time for membrane to get larger or replace lost lipids the smooth ER synthesizes new lipids using machinery located on the cytosolic surface