Midterm No. 2, Opus 3

Question 1

What is the role of caspases (a type of protease) during apoptosis?

Answer 1

They inactivate flippases and activate scramblases

The activated scramblases flip PS to the exoplasmic side. The inactivated flippases are unable to reverse this.

Question 2

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?

Answer 2

Also in the cytosol

Question 3

How much of the human proteome is directed to the ER?

Answer 3

1/3

Question 4

Why is folding and quality control in the ER difficult?

Answer 4

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)

Question 5

Who helps with ER quality control? (list 4)

Answer 5

Chaperones (members of the Hsp70, Hsp90, and Hsp101 superfamilies)

Specialized folding enzymes

Regulators of entry from the ER to the golgi

Degradation factors

Question 6

How does BiP promote proper folding in the ER lumen?

Answer 6

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)

Question 7

What are lectins (general answer)?

Answer 7

Carbohydrate binding proteins

Question 8

Calnexin (CNX) and calreticulin (CRT)

Answer 8

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

Question 9

What recognizes overly processed N-glycosylation?

Answer 9

OS-9

It targets the protein for degradation

Question 10

What recognizes N-glycosylation that isn’t fully processed?

Answer 10

Calnexin (CNX) and calreticulin (CRT)

Question 11

OS-9

Answer 11

Recognizes overly processed N-glycosylation (specifically too few mannoses) and targets the protein for degradation

Question 12

ER Associated Degradation (ERAD)

Answer 12

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

Question 13

Unfolded protein response

Answer 13

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

Question 14

What types of proteins recognizes unfolded and/or misprocessed proteins in the ERAD system?

Answer 14

Hsp70 chaperones, ER lectins, or some other factors in the ER lumen

Question 15

Retrotranslocation

Answer 15

The “spitting out” / removal of unfixable proteins from the ER (to be moved to and degraded in a proteosome)

Question 16

Quickly list 3 examples of unfolded protein responses

Answer 16

Ire1, PERK, and ATF6

Question 17

Ire1 unfolded protein response

Answer 17

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

Question 18

Where is BiP in the Ire1 unfolded protein response when cells are happy?

Answer 18

Bound to Ire1

Exists as an Ire1+BiP monomer

Question 19

Where is BiP in the Ire1 unfolded protein response when cells are unhappy?

Answer 19

BiP is busy

Ire1 homodimerizes and autophosphorylates to Ire1+Ire1

Question 20

PERK unfolded protein response

Answer 20

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)

Question 21

Where is BiP in the PERK unfolded protein response when cells are happy?

Answer 21

BiP is bound to PERK

Exists as a PERK+BiP monomer

Question 22

Where is BiP in the PERK unfolded protein response when cells are unhappy?

Answer 22

BiP is busy

PERK homodimerizes and autophosphorylates to PERK+PERK

Question 23

What is the importance of ATF4 in the unfolded protein repsonse?

Answer 23

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)

Question 24

ATF6 unfolded protein response

Answer 24

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

Question 25

Where is BiP in the ATF6 unfolded protein response when cells are happy?

Answer 25

BiP is bound to ATF6 as a homodimer, secured with disulfide bonds BiP-ATF6+Bip-ATF6

Question 26

Where is BiP in the ATF6 unfolded protein response when cells are unhappy?

Answer 26

BiP is busy The dimer is reduced, disulfide bonds are broken. ATF6 is now a monomer

Question 27

Symptoms of cystic fibrosis

Answer 27

Excessive buildup of mucus in the lungs and intestines, and very salty skin

Question 28

Cause of cystic fibrosis

Answer 28

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

Question 29

Most common mutation that causes cystic fibrosis

Answer 29

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.

Question 30

If F508del variants are partially functional, can such patients be helped by inhibiting ERAD?

Answer 30

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

Question 31

How thick are average lipid bilayers?

Answer 31

~5 nm Twice as big as a DNA double helix (2 nm)

Question 32

Do sphingolipids use glycerol?

Answer 32

No

Question 33

What functional group is found on a sphingolipids 3 carbon linker?

Answer 33

An amino group

Question 34

What types of lipids are membrane rafts often enriched with?

Answer 34

Sphingolipids and cholesterol

Question 35

True or false: plants have sterols, but lack cholesterol

Answer 35

True

Question 36

What self-sealing lipid structure is common to detergents?

Answer 36

Micelles

Question 37

How do thermophilic archaea (remember those funky lil fuckers?) link their glycerol to their hydrophobic membrane moieties?

Answer 37

Ether linkages. These are more thermally stable and resist degradation at high temperatures Note that bacteria (and probably everything else) use ester linkages

Question 38

What molecule do thermophilic archaea use for their hydrophobic membrane moieties?

Answer 38

Phytanyls They are subunits of isoprene, and are not fatty acids!

Question 39

What unique structural features do thermophilic archaea use to prevent their membranes from degrading at high temperatures (adaptations to extremely hot environments?)

Answer 39

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

Question 40

Bacteriorhodopsin

Answer 40

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

Question 41

How does a phospholipid’s head group contribute to overall lipid shape?

Answer 41

Larger head groups make the entire molecule cylinder shaped Smaller head groups make the entire molecule cone shaped (head is the point)

Question 42

How does a phospholipid’s head group contribute to membrane curvature?

Answer 42

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)

Question 43

How do you mathematically represent how a protein’s size affects its mobility (diffusion) through a membrane?

Answer 43

D = (k T) / (6 pi n R)

Question 44

For the following equation: D = (k T) / (6 pi n R) What is D?

Answer 44

Diffusion constant Numerical representation of a protein’s mobility within a membrane

Question 45

For the following equation: D = (k T) / (6 pi n R) What is k?

Answer 45

Boltzmann constant

Question 46

For the following equation: D = (k T) / (6 pi n R) What is T?

Answer 46

Temperature

Question 47

For the following equation: D = (k T) / (6 pi n R) What is n?

Answer 47

Viscosity of the membrane (numerical value)

Question 48

For the following equation: D = (k T) / (6 pi n R) What is R?

Answer 48

Radius of the protein (or whatever particle in question)

Question 49

What is the function of cell barriers?

Answer 49

Maintains distinct zones between sections of a cell’s plasma membrane

Question 50

What’s the difference between F-class and V-class ATP synthases?

Answer 50

V-class pumps spend ATP to create a H+ gradient F-class pumps use H+ gradients to make ATP

Question 51

True or false: the ER membrane is continuous within the nuclear envelope

Answer 51

True

Question 52

True or false: the ER is completely separate from the nucleus

Answer 52

False Their membranes are continuous

Question 53

Are proteins fully folded when entering the nucleus?

Answer 53

Yes

Question 54

Are proteins fully folded when exiting the nucleus?

Answer 54

Yes

Question 55

How many proteins are NPCs made of?

Answer 55

500 to 1000

Question 56

How large are NPCs?

Answer 56

Around 110 nm in diameter, weighs around 125 million daltons

Question 57

How many NPCs can be found in the average cell?

Answer 57

3k to 4k

Question 58

What substances/molecules are completely permeable to the NPC’s central channel?

Answer 58

Ions, ATP, sugars, and anything else less than 5 kDa

Question 59

What substances/molecules are partially permeable to the NPC’s central channel?

Answer 59

All molecules between 5 and 40 kDa

Question 60

What substances/molecules are not at all permeable to the NPC’s central channel?

Answer 60

Anything larger than 40 kDa, so large proteins and protein complexes

Question 61

Can membrane proteins in the NPC move freely between the inner and outer membrane?

Answer 61

No, they are bound in the NPC

Question 62

What makes up the NPC’s central channel?

Answer 62

Flexible gel-like mesh matrix of FG-nucleoporins

Question 63

What are FG-nucleoporins?

Answer 63

The free ends of the NPCs central channel proteins. They are mostly extend IDRs with some phenylalanine-glycine (FG) repeats interspersed throughout

Question 64

How do proteins larger than 40 kDa enter the nucleus?

Answer 64

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

Question 65

How do proteins larger than 40 kDa exit the nucleus?

Answer 65

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

Question 66

How do proteins smaller than 40 kDa enter the nucleus?

Answer 66

They passively diffuse across the NPCs mesh of FG-nucleoporins

Question 67

How do proteins smaller than 40 kDa exit the nucleus?

Answer 67

They passively diffuse across the NPCs mesh of FG-nucleoporins

Question 68

Are GEFs kinases?

Answer 68

NO!

Question 69

Are GAPs phosphatases?

Answer 69

NO!

Question 70

What does RanGTP do during import?

Answer 70

It disassembles import complexes

Question 71

What does RanGTP do during export?

Answer 71

It assembles the exportin complex

Question 72

How do RanGTP levels remain high in the nucleus?

Answer 72

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

Question 73

What are the two general types of experimental designs?

Answer 73

Necessary and sufficient

Question 74

Where is the information for protein folding found?

Answer 74

Inside the protein itself (primary sequence)

Question 75

Where is the information for protein localization/targeting found?

Answer 75

Inside the protein itself (mostly primary sequences, but sometimes tertiary 3D shapes)

Question 76

What does GAP do?

Answer 76

GTP –> GDP Hydrolyzes, removes a phosphate

Question 77

What does GEF do?

Answer 77

Kinase, GDP --> GTP