Midterm No. 2, Opus 3 Flashcards
What is the role of caspases (a type of protease) during apoptosis?
They inactivate flippases and activate scramblases
The activated scramblases flip PS to the exoplasmic side. The inactivated flippases are unable to reverse this.
If an integral membrane protein in the ER membrane has its C-terminus is in the cytosol, where will its C-terminus be once it’s moved to the plasma membrane?
Also in the cytosol
How much of the human proteome is directed to the ER?
1/3
Why is folding and quality control in the ER difficult?
Because the ER has a high concentration of unfolded proteins, a high concentration of Ca2+, and it’s an oxidizing environment
(the oxidizing environment is necessary for the disulfide bonds to form, but it’s a bit dangerous for unfolded proteins as it incentivizes mutation and misfolding. This is why we worry about reactive oxygen species)
Who helps with ER quality control? (list 4)
Chaperones (members of the Hsp70, Hsp90, and Hsp101 superfamilies)
Specialized folding enzymes
Regulators of entry from the ER to the golgi
Degradation factors
How does BiP promote proper folding in the ER lumen?
It binds to the more vulnerable regions of a nascent proteins, like moderately hydrophobic patches
It protects regions of around 7-9 amino acids long, and prevents premature folding (folding before synthesis is complete)
What are lectins (general answer)?
Carbohydrate binding proteins
Calnexin (CNX) and calreticulin (CRT)
CNX and CRT play analogous roles in quality checking N-linked glycosylation
They recognize N-glycosylation that isn’t fully processed yet, and keep the protein in the ER to finish the processing
What recognizes overly processed N-glycosylation?
OS-9
It targets the protein for degradation
What recognizes N-glycosylation that isn’t fully processed?
Calnexin (CNX) and calreticulin (CRT)
OS-9
Recognizes overly processed N-glycosylation (specifically too few mannoses) and targets the protein for degradation
ER Associated Degradation (ERAD)
Mechanism to eject misfolded and/or misprocessed proteins from the ER to be destroyed by cytosolic proteosomes
If the target proteins can’t be fixed, improperly folded proteins are exported from the ER and degraded in the cytosol
The problems are recognized the ER Hsp70 chaperones, ER lectins, or some other factors inside the ER
A Protein Translocator Complex (PTC) does retrotranslation, which moves unfixable proteins from the ER lumen outside to the cytosol
The PTC works alongside an E3 ubiquitin ligase, which polyubiquitinates the bad protein as it exits the PTC. (Note that the E3 ubiquitin ligase is ATP dependent, ATP→ADP+Pi)
There are NO proteasomes inside the ER lumen! To be degraded, proteins must be retrotranslocated!
The spitting out is retrotranslocation. The entire processes is ERAD
ERAD is a very selective process. Local process, single proteins. It’s not an emergency alarm
Unfolded protein response
Mechanisms to shut down general translation and upregulate chaperones and other protective proteins in response to stress using BiP as the signal/sensor
When cells are happy, BiP is found bound to some ER membrane protein.
When cells are unhappy there are tons of unfolded proteins in the ER lumen, BiP is busy dealing with the unfolded proteins and leaves the membrane protein. This causes a conformational change in the membrane protein, which will have some downstream effect on transcription and/or translation
This is a global response. It happens during stress and other big problems, situations that cause mass protein unfolding
BiP’s absence is the sensor that triggers this response
Summary: when BiP is busy, that means there’s a lot of unfolded proteins in the ER, and indication of stress. In response, general translation is shut down and transcription of specific proteins to mitigate the stress is activated. This is emergency mode. Resources need to be spent on resolving the emergency, not on normal cell activities.
The different unfolded protein responses happen simultaneously
What types of proteins recognizes unfolded and/or misprocessed proteins in the ERAD system?
Hsp70 chaperones, ER lectins, or some other factors in the ER lumen
Retrotranslocation
The “spitting out” / removal of unfixable proteins from the ER (to be moved to and degraded in a proteosome)
Quickly list 3 examples of unfolded protein responses
Ire1, PERK, and ATF6
Ire1 unfolded protein response
Ire1 is an ER membrane protein
When cells are happy, it’s bound to BiP and exists as a BiP+Ire1 monomer
When cells are unhappy and BiP is busy, Ire1 homodomerizes and autophosphorylates → Ire1+Ire1
In dimer form, Ire1+Ire1 can now function as an endonuclease. It cuts and splices mRNA
If it cuts an Hac1 mRNA (aka an XPB1 mRNA), the cut jumpstarts self-splicing of the mRNA. The fully spliced transcript is then translated into an Hac1 transcription factor (aka an XPB1 TF), which enters the nucleus and activates transcription of ER chaperones, ER quality control factors, and lipid synthesis factors
Where is BiP in the Ire1 unfolded protein response when cells are happy?
Bound to Ire1
Exists as an Ire1+BiP monomer
Where is BiP in the Ire1 unfolded protein response when cells are unhappy?
BiP is busy
Ire1 homodimerizes and autophosphorylates to Ire1+Ire1
PERK unfolded protein response
PERK exists as a monomer when BiP is bound
When BiP is busy, PERK autophosphorylates and homodimerizes to PERK+PERK
The dimer then phosphorylates eIF2alpha, which blocks the eIF2alpha from helping the small ribosomal subunit bind to specialized (charged) tRNAs. The phosphorylated eIF2alpha blocks a lot of translation
However there are some mRNAs that can be translated in a variant way that doesn’t need eIF2alpha. One such gene is ATF4, which once translated, enters the nucleus and acts as a TF for redox enzymes, GADD34, and CHOP (all things involved in mitigating cell stress)
Where is BiP in the PERK unfolded protein response when cells are happy?
BiP is bound to PERK
Exists as a PERK+BiP monomer
Where is BiP in the PERK unfolded protein response when cells are unhappy?
BiP is busy
PERK homodimerizes and autophosphorylates to PERK+PERK
What is the importance of ATF4 in the unfolded protein repsonse?
It’s part of the PERK unfolded protein response
It can be translated without eIF2alpha, so it can be translated even when eIF2alpha is blocked during cell stress
Once translated it enters the nucleus and becomes a TF for redox enzymes, GADD34, and CHOP (all things involved in mitigating cell stress)
ATF6 unfolded protein response
When BiP is bound ATF6 is found as a homodimer, bound together with disulfide bonds
When BiP is busy, the dimer is reduced and the disulfide bonds are broken. ATF6+ATF6 breaks down into monomers
The ATF6 monomers are then trafficked from the ER membrane to the golgi (note: in the diagram, the ER lumen head of the ATF6 monomer is shown in the golgi lumen while the cytosol tail remains in the cytosol for both locations)
The ATF6 monomer is processed (cut) in the golgi to become an ATF6 TF, which then enters the nucleus and promotes transcription of ER chaperones, ER quality control factors, and XPB1. This creates a feedback pattern
Where is BiP in the ATF6 unfolded protein response when cells are happy?
BiP is bound to ATF6 as a homodimer, secured with disulfide bonds
BiP-ATF6+Bip-ATF6
Where is BiP in the ATF6 unfolded protein response when cells are unhappy?
BiP is busy
The dimer is reduced, disulfide bonds are broken. ATF6 is now a monomer
Symptoms of cystic fibrosis
Excessive buildup of mucus in the lungs and intestines, and very salty skin
Cause of cystic fibrosis
Defective CFTR, an ABC transporter that exports chloride ions out of cells. The defect causes a buildup of Cl- ions inside the cells, so the cells import more Na+ to mitigate the negative charge, which then forms into salt and causes the cell to import more H2O. This cycle effectively dries any extracellular fluids or mucus, preventing it from clearing out as usual and causing a thick, sticky buildup. It’s a cascade of problems
Most common mutation that causes cystic fibrosis
F508del
This deletion slows the folding of the CFTR as it is being synthesized, causing it to be degraded before it can leave the ER. the CFTR becomes a victim of the ERAD system.
If F508del variants are partially functional, can such patients be helped by inhibiting ERAD?
Yes
There are new drugs on the market that have been super beneficial to partial F508del cystic fibrosis patients that inhibit ERAD as their action mechanism
How thick are average lipid bilayers?
~5 nm
Twice as big as a DNA double helix (2 nm)
Do sphingolipids use glycerol?
No
What functional group is found on a sphingolipids 3 carbon linker?
An amino group
What types of lipids are membrane rafts often enriched with?
Sphingolipids and cholesterol
True or false: plants have sterols, but lack cholesterol
True
What self-sealing lipid structure is common to detergents?
Micelles
How do thermophilic archaea (remember those funky lil fuckers?) link their glycerol to their hydrophobic membrane moieties?
Ether linkages. These are more thermally stable and resist degradation at high temperatures
Note that bacteria (and probably everything else) use ester linkages
What molecule do thermophilic archaea use for their hydrophobic membrane moieties?
Phytanyls
They are subunits of isoprene, and are not fatty acids!
What unique structural features do thermophilic archaea use to prevent their membranes from degrading at high temperatures (adaptations to extremely hot environments?)
Biphytanyl/lipid monolayer. Hydrophobic domains are covalently linked, forming a monolayer
Some have cyclopentane rings in the monolayer
Some use branched lipid chains in their membranes
Bacteriorhodopsin
An example of an integral membrane protein
Has multiple alpha-helix TMDs, forms a channel through the plasma membrane
Halophilic mixotrophs can use bacteriorhodopsin to switch between nutritional states
Other species use it to pump Cl- and H+ ions out in response to light
How does a phospholipid’s head group contribute to overall lipid shape?
Larger head groups make the entire molecule cylinder shaped
Smaller head groups make the entire molecule cone shaped (head is the point)
How does a phospholipid’s head group contribute to membrane curvature?
The cytosolic leaflets of curved membranes is made up of cone shaped lipids (lipids with small head groups)
The exoplasmic leaflet of curved membranes is made of the larger cylinder shaped lipids (lipids with large head groups)
How do you mathematically represent how a protein’s size affects its mobility (diffusion) through a membrane?
D = (k T) / (6 pi n R)
For the following equation:
D = (k T) / (6 pi n R)
What is D?
Diffusion constant
Numerical representation of a protein’s mobility within a membrane
For the following equation:
D = (k T) / (6 pi n R)
What is k?
Boltzmann constant
For the following equation:
D = (k T) / (6 pi n R)
What is T?
Temperature
For the following equation:
D = (k T) / (6 pi n R)
What is n?
Viscosity of the membrane (numerical value)
For the following equation:
D = (k T) / (6 pi n R)
What is R?
Radius of the protein (or whatever particle in question)
What is the function of cell barriers?
Maintains distinct zones between sections of a cell’s plasma membrane
What’s the difference between F-class and V-class ATP synthases?
V-class pumps spend ATP to create a H+ gradient
F-class pumps use H+ gradients to make ATP
True or false: the ER membrane is continuous within the nuclear envelope
True
True or false: the ER is completely separate from the nucleus
False
Their membranes are continuous
Are proteins fully folded when entering the nucleus?
Yes
Are proteins fully folded when exiting the nucleus?
Yes
How many proteins are NPCs made of?
500 to 1000
How large are NPCs?
Around 110 nm in diameter, weighs around 125 million daltons
How many NPCs can be found in the average cell?
3k to 4k
What substances/molecules are completely permeable to the NPC’s central channel?
Ions, ATP, sugars, and anything else less than 5 kDa
What substances/molecules are partially permeable to the NPC’s central channel?
All molecules between 5 and 40 kDa
What substances/molecules are not at all permeable to the NPC’s central channel?
Anything larger than 40 kDa, so large proteins and protein complexes
Can membrane proteins in the NPC move freely between the inner and outer membrane?
No, they are bound in the NPC
What makes up the NPC’s central channel?
Flexible gel-like mesh matrix of FG-nucleoporins
What are FG-nucleoporins?
The free ends of the NPCs central channel proteins. They are mostly extend IDRs with some phenylalanine-glycine (FG) repeats interspersed throughout
How do proteins larger than 40 kDa enter the nucleus?
They are escorted by importin
Importin binds to the cargo and brings it through the NPC
Inside the nucleus, GEF phosphorylates RanGDP to RanGTP
RanGTP replaces the cargo and binds to importin. They both then exit the nucleus
Once back in the cytosol, RanGTP is hydrolyzed by GAP to RanGDP
How do proteins larger than 40 kDa exit the nucleus?
They are escorted by exportin
Inside the nucleus, cargo is bound to exportin and RanGTP.
They exit the nucleus through an NPC together
Once in the cytosol, RanGTP is hydrolyzed by GAP to RanGDP.
This causes the cargo to disassociate from exportin
Exportin and RanGDP then re-enter the nucleus
RanGDP must be phosphorylated by GEF to become RanGTP and begin again
How do proteins smaller than 40 kDa enter the nucleus?
They passively diffuse across the NPCs mesh of FG-nucleoporins
How do proteins smaller than 40 kDa exit the nucleus?
They passively diffuse across the NPCs mesh of FG-nucleoporins
Are GEFs kinases?
NO!
Are GAPs phosphatases?
NO!
What does RanGTP do during import?
It disassembles import complexes
What does RanGTP do during export?
It assembles the exportin complex
How do RanGTP levels remain high in the nucleus?
Localized GAPs and GEFs phosphorylate and hydrolyze things as necessary to keep RanGTP levels high in the nucleus
GAPs are bound to the NPC’s cytoplasmic filaments
GEFs are bound to the nearby chromatin inside the nucleus
What are the two general types of experimental designs?
Necessary and sufficient
Where is the information for protein folding found?
Inside the protein itself (primary sequence)
Where is the information for protein localization/targeting found?
Inside the protein itself (mostly primary sequences, but sometimes tertiary 3D shapes)
What does GAP do?
GTP –> GDP
Hydrolyzes, removes a phosphate
What does GEF do?
Kinase, GDP –> GTP
