ER & Golgi Flashcards
What part of ER has ribosome receptors?
Rough ER
What is the function of sER?
lipid production & detoxification
sterod hormone synthesis
muscle contraction & relaxation
glycogen degradation & gluconeogenesis
The rER is ____ with nuclear membrane
continuous
Soluble proteins are ____
excreted
Transmembrane proteins become part of ______
plasma membrane
What is the function of the rER?
proteins destined for ER, golgi, endosomes, lysosomes, plasma membrane & secretion
helps fold proteins
monitors assembly, retention, and degradation of proteins
What cells would have a lot of sER?
adrenal cortex, cells of testes (leydig) - they need to make lots of hormones
hepatocyte sER makes what detoxification enzyme?
Cytochrome P450
What is the purpose of Glucose-6-phosphatase and where is it located?
removes phosphate from G6P, located in sER
If a person is taking medication what might increase in cells to help detoxify?
sER
Where are proteins synthesized?
Cytosol, unless they have signal sequence that brings them to rER. Otherwise they are translated on ribosome in cytoplasm.
Once mRNA leave nucleus it always combines with ____ _____
free ribosome
What are the two ways a protein can be taken from cytoplasm into organelle?
co-translational translocation
post-translational translocation
What is co-translational translocation?
While a protein is being translated it is transported to rER
What is post-translational translocation?
After a protein is made a signal sequence allows it to be transported to an organelle
What does SRP stand for?
Signal recognition particle
What is an ER signal sequence and where is it found?
A sequence at the N-terminus of protein, signaling for it to be moved to ER. It signals to an SRP
What is the purpose of SRP?
it binds to the ER signal sequence and brings the ribosome to a translocon and transfers it, then is displaced
Describe all the steps in cotranslational translocation
(pg. 96 in ppt)
1. ER signal sequence on protein
2. SRP binds to signal sequence
3. SRP brings protein over to translocation channel
4. SRP detaches
5. Ribosome transfers growing polypeptide chain through ER membrane at the translocon
What is a translocon?
A pore complex on membrane of ER that the growing protein goes through
What does BiP stand for?
Binding protein
What will always happen to the N-terminal signal sequence?
It will be cleaved off
What cleaves the ER sequence?
signal peptidase
What is the purpose of BiP?
helps proteins fold, it binds the peptide in the ER lumen and pulls it in
What does it mean that a BiP is a lumeneal ER chaperone?
It helps proteins fold
What is a single-pass transmembrane protein?
a protein that has a stretch of hydrophobic amino acids and stays in the ER membrane, the rest of protein is translated in cytoplasm
Describe the process of a single-pass transmembrane protein being translated
ER terminal sequence (usually N-terminus) - translocation to rER, a hydrophobic sequence in protein anchors it to ER membrane as a STOP sequence, the rest is translated in cytoplasm. When translation complete, N terminal cleaved, but it remains in membrane b/c of hydrophobic region
How does the hydrophobic region of a translocating protein fold?
alpha helical structure
What is an internal signal sequence?
It is a signal sequence (like ER signal sequence) that is not on the end of a protein - it’s in the middle, and it initiates translocation
Does a single-pass transmembrane protein with internal signal sequence enter cytoplasm with C or N terminus?
can be either, depends on sequence
If the signal sequence is internal, is it still cleaved?
No
What is a multipass transmembrane protein?
It is a protein that has multiple start and stop sequences and is “stitched” into plasma membrane. It’s basically a transmembrane protein with many parts in the membrane (the parts that cross the membrane are alpha-helices)
What is PDI?
Protein disulfide isomerase
What are chaperones?
They recognize and bind unfolded or misfolded protein, helps fold correctly
Name 3 ER resident proteins
BiP, Calnexin, Calreticulin
What does a PDI do?
it helps reaction - oxidation of free SH groups on cysteins (S-S bonds) - helps fold protein
Once protein is made in the rER, what happens?
disulfide bonds, protein is modified - needs proper folding, addition of carbohydrates, proteolytic cleavage, oligomerisation
Cis golgi receives proteins from the ____
ER
What is the function of trans golgi?
sorts and packages proteins into vesicles to go to plasma membrane
What happens to proteins if they aren’t folded correctly?
They are polyubiquinated - ultimate degradation
Where are sugar chains modified?
golgi
Sugar chains are added to protein om ER. What is the last sugar added?
Glucose (3 glucose)
How are proteins glycosylated?
Dolichol assembles the carbohydrates and puts transfers them to protein. They are added to N terminus of Asn residues. Total of 14 sugars
Where are sugars attached to protein in ER?
N-termins of Asn
What is dolichol?
a membrane bound lipid that assembles and transfers sugars (oligosaccharide) to protein
What happens to protein and sugar once it is properly folded?
Last four sugars are cleaved off - 3 glucose and a mannose. This is signal that it is correctly folded
where are the 4 sugars cleaved off of a properly folded protein?
ER
Where do you get specificity of sugar chains?
golgi
In golgi, there is more N-linked trimming and/or addition of ______
monosaccharides
What is O-linked glycosylation?
when sugars are attached to OH of serine and threonine
What does GPI stand for?
glycosylphosphatidyl-inositol
Where are GPI linked proteins assembled?
ER
GPI linked proteins are commonly found in ___ rafts
lipid
Describe what happens with GPI anchors when a protein is destined for a plasma membrane
It is covalently attached to GPI anchor in ER, then usually directs protein to lipid raft
If a protein will not fold correctly in the ER, what happens to it?
Ejected via translocon to cytosol, where it is deglycosyalated & ubiquilated, taken up by proteasome and degraded
key words: CFTR mutation, fibrosis in lungs, sweat, autosomal recessive, respiratory failure are of what disease?
Cystic Fibrosis
What is a Pseudomona?
opportunistic infection meaning that some underlying condition or immunosuppression is affecting pt
What is the mode of inheritance for cystic fibrosis?
Autosomal recessive
What disease are these symptoms from? young white kid; normal at birth, failure to thrive, recurrent pneumonia, sputum +pseudomonas; PE: scattered rales/crackles throughout lungs, rhonchi; diagnostic: axillary sweat test; bronchiectasis; respiratory failure is most common cause of death
Cystic Fibrosis
What is the mechanism for cystic fibrosis?
CFTR is misfolded in the ER so it keeps getting ejected back to cytosol and degraded in proteasomes
What is CFTR?
A protein that is a channel for the movement of Cl- ions across cell
What would happen if CFTR protein was transported to plasma membrane?
It would function normally - but because it folds incorrectly the body polyubiquinates it
What is Bronchiectasis?
can develop due to CF. It is a scarring of the lung field.
What causes bronchiectasis?
chronic infections. they lead to inflammation and scarring
Bronchiectasis leads to _____ bronchial dilation and thickened ____ walls
irreversible, bronchial
What is the most common cause of death in CF pts?
respiratory failure
What is Hypercholesterolemia?
familial high cholesterol
These key words: LDL-R, CHD (CAD), plasma cholesterol, Xanthomata, Xanthelasmata are what disease?
familial high cholesterol
these symptoms are what disease? increased plasma cholesterol levels; increased LDL synthesis; major risk factor for CHD, premature atherosclerosis, Xanthomata, corneal arcus, Xanthelasmata
Hypercholesterolemia
What is the mechanism for hypercholesterolemia?
Mutation inhibits proper folding of LDL-R ! ejected from ER, polyubiquinated, degraded by proteasome
LDL receptor isn’t properly folded. Normally it binds to LDL and brings it in to be broken down and used. This defect means LDL is just circulating and accumualating in vessels and skin
Does the LDL protein ever leave the membrane?
No - the membrane itself creates the vesicle and brings it to cell membrane
Describe what happens to a normal person without Familial hypercholesterolemia
This diagram is what normall yhappens: cell needs to pick up LDL, needs LDL receptor (transmembrane proteins), goes to ER, needs to fold properly, when folded correctly goes to golgi, protein undergoes various posttranslational modifications (glycosylations), vesicle to plasma membrane and LDL receptor is now in plasma membrane. The protein in membrane is never leaving the membrane, the membrane itself is what is leaving and going to golgi, plasma membrane. The protein itself doesn’t move.
The LDL receptors on surface can now pick up LDL (low density lipid particles). LDL receptors combine to LDL – triggers endocytosis, invaginate and vesicle enters cell bringing LDL bound to receptor. LDL receptors recycled back to plasma membrane. LDL delivered to lysosome, hydrolytic enzymes break it down. Cholesterol esters can now be used. If cell has enough cholesterol it inhibits production of more LDL receptors and enzyme HMG coA reductase (enzyme that synthesize cholesterol).
What is CHD?
coronary heart disease
Pts with Familial hypercholesterolemia are at major risk for what?
CHD
If the ER and golgi membrane pinch off with the proteins, how do they not eventually disappear?
recycling of membrane
What is the roe of microtubules in vesicle transport?
The vesicles are walked along the microtubules to their destination via a motor protein
What is COPII?
It coats the membrane where a vesicle is forming and budding off. Once the vesicle has left the membrane COPII disassembles
Do motor proteins walk both directions down the microtubule?
No, because microtubules have a + and - side, motor proteins will walk a certain direction
What is retrieval transport?
Recycles membrane, and puts proteins that are in the wrong spot back in the right spot
If a protein doesn’t have a signal sequence, where will it be translated?
In cytoplasm
If a protein has a NLS signal sequence where will it be translated?
Nucleus
If a protein has a SKL signal sequence where will it be translated?
perixisome
What is the pathway for a protein with an ER signal sequence only?
It will finish translation in rER, to golgi, to plasma membrane. It will be secreted once at the plasma membrane, unless it is hydrophobic, then it will be a transmembrane protein
If a protein has a KDEL signal sequence, where will it be translated?
KDEL is a retrieval sequence. It will have KDEL sequence in addition to an ER signal sequence. The KDEL is recognized and trigger COPII formation, which elicts retrieval of protein back to ER
If a protein has a KKXX sequence, where will it be translated?
ER - this is same mechanism as KDEL sequence. The X just stands for any amino acid (I think)
If a protein has an ER signal sequence and then aquires an M6P, where will it be translated?
It is translated in ER, then in golgi then golgi phosphorylates it. There are M6P receptors that recognize the sequence and take it to its finally destination, the lysosome
What is M6P?
It is phosphorylation of a protein. This occurs after protein has been translated and moves to the golgi - the golgi phosphorylates it. M6P is a tag that lets the cel know it needs to be moved to the lysosome
If the N terminus is in the lumen of the ER when it is being translated, where will it be once it joins the plasma membrane?
The N-terminus will face the extra-cellular space if it is hydrophobic. If hydrophilic the entire thing will be secreted
What happens on trans-golgi?
It buds and sends proteins to the plasma membrane
What is the ERGIC and what does it stand for?
ER-Golgi Intermediate compartment. It contains vesicular tubular clusters
What side is cis golgi facing?
It is facing the rER
What does CGN stand for?
cis golgi network
Vesicles that are leaving need to be coated in COP protein except for where?
vesicles budding off of trans golgi do not need COP coat
Proteins with KDEL or KKXX tags are ___ resident proteins
ER
How are proteins that end up in golgi by mistake brought back to ER?
Via COPI
How are proteins in ER brought to golgi?
Via vesicles, need to be coated in COPII
What is COPI?
It creates a coat to make a vesicle around proteins that are ER resident proteins but are in the golgi. It helps bring them back
What is COPII?
It creates a coat to make a vesicle around proteins that are in the RER and need to be brought to Cis golgi
Is KDEL hydrophobic or hydrophilic?
Hydrophilic, it’s soluble
Is KKXX hydrophobic or hydrophillic?
hydrophobic
How does COPI bring back KDEL if it mistakenly enters golgi?
KDEL is soluble, and COPI can’t bind to it b/c it’s soluble. There is a KDEL receptor in membrane of golgi, KDEL binds to it. The binding causes a conformational change, and then COPI binds to the receptor and carries it back to the ER
How does COPI bring back KKXX if it mistakenly enters golgi?
Because KKXX is hydrophobic, COPI can bind directly to it. It binds and carries it back to the ER
What are ARF/SAR-GTP
G proteins
What is ARF/SAR-GTP function?
they are involved in COP coat formation to make vesicle
What binds to soluble proteins so they can leave membrane of ER?
Cargo receptors
What needs to happen to ARF/SAR for them to help form COP assembly?
They need to be GTP bound
Once the vesicle breaks off from the membrane, what happens to the COP coat?
It is disassembled
What causes the COP coat to disassemble after it buds off from membrane?
GTP is hydrolyzed to GDP, so hydrophobic amino aids flip out and COP coat is disassembled
Why does the COP coat need to disassemble before the vesicle reaches its destination?
so motor proteins can carry the vesicle, so other proteins can interact for vesicle fusion with target membrane
What happens once the COP coat disassembles on vesicle?
motor proteins bind to vesicle and carry it to target membrane
How do carrier proteins get vesicle to target?
They “walk” along microtubules
What is Rab?
G-protein
What does V-snare stand for?
vesicle snare
What does T-snare stand for?
target membrane snare
Where is V snare located?
On vesicle
What state is Rab in on the vesicle?
GTP bound
What is the purpose of the Rab on the vesicle?
It interacts with another Rab or a Rab binding protein on the membrane and brings the vesicle close enough to the membrane for V & T snares to interact
After the Rab and Rab binding proteins bind, what happens?
v-snare on vesicle interacts with t-snare on memrane and brings the vesicle close enough to fuse
After the membrane is fused, what happens to V-snare and T-snare
a complex of proteins disentangles them, then t-snare stays and v-snare is recycled back to ER
What part do v & t snare play in ensuring specificity?
If the proteins are supposed to go to golgi, their v-snares will only be compatible with t-snares on golgi. Same if it was going to lysosome, etc.
If a cell is secreting a lot of protein, it will have a lot of what two things?
ER & Golgi
What are the two transport models for how vesicles move through the golgi?
Vesicular transport model & Cisternal Transport model
Describe the vesicular transport model
Each stack of golgi is stationary. Protein goes to cis golgi and leaves via vesicle and fuse with the next membrane stack. Resident enzymes act on it, modifies, then it buds off in a vesicle and goes to next stack, where resident enzymes modify it. Ultimately to trans golgi and to plasma membrane. They are transferred via a vesicle to each stack.
Describe the cisternal transport model
Cisternal maturation model – vesicles coming off of ER will all fuse together to create a new cis golgi. Enzymes that were present in previous cis golgi will be moved into newly forming cis golgi. So the cis golgi will ultimately mature and become a stack farther up closer to trans. Enzymes move from the previous cis to the newly forming one.
Where does the protein receive its M6P tag destining it for the lysosome?
In the cis golgi
If a protein needs to go to lysosom, what will it be tagged with?
M6P
Phosphorylation of what makes M6P?
mannose
What specifically puts the tag on the protein in the golgi for it to go to lysosome?
N-acetylglucosamine phosphotransferase
What scenario allows a protein to end up in the cytoplasm?
No tags at all, or polyubiquinated (because its folded improperly)
Name an amino acid is involved in N-linked glycosylation?
Asparagine (pt 131)
Name an amino acid involved in O-linked glycosylation
Threonine (pg 131)
A deficiency of N-acetylglucosamine phosphotransferase leads to what disease?
I-cell disease
what is another name for I-cell disease?
Mucolipidosis II
What is the mode of inheritance for I-cell disease?
Autosomal recessive
key words: absence of M6P, waste products in inclusion bodies, coarse facial features, enlarged liver/spleen, death CHF are what disease?
I-cell disease
What is the mechanism for I-cell disease?
Lysosomal enzymes do not receive the M6P tag. Specifically, there is a loss of function of N-acetylglucosamine phosphotransferase, which puts on the M6P tag.. There isn’t anything wrong with the enzymes themselves, they just can’t be sorted correctly. Ultimately, because the proper enzymes aren’t getting to lysosome, there is an accumulation of non-digested things in lysosome. Waste products accumulate as inclusion bodies
symptoms: progressive disease; high levels of acid hydrolases in the blood; skeletal abnormalities, coarse facial features, restricted joint movement, psychomotor retardation, enlarged spleen, liver, & heart valves, death due to CHF or RTI are what disease?
I-cell disease
What is the life expectancy of a pt with I-cell disease?
<10 years
Where is the mutation located in cystic fibrosis?
Delta F508
What class is familial hypercholesterolemia where defective transport from rER to Golgi (don’t reach PM
Class II
