Week 3: Protein Sorting

Question 1

symbiotic theory

Answer 1

mitochondria were an invasive bacterial species and were engulfed by an archaeon by the expansion of its membrane
- protrusions fused with one another to pinch off internal membrane-enclosed compartments

Question 2

which compartments are topologically equivalent

Answer 2

1. the nucleus and the cytosol
2. the perinuclear space and the rest of the endomembrane system with the extracellular area

Question 3

what is a condensate?

Answer 3

a scaffold of macromolecules not enclosed by a membrane that is made of protein and RNA as well as the client molecules they attract

Question 4

describe one situation where a condensate can form and dissolve again

Answer 4

in class the example was phosphorylation of a receptor on a membrane. When the ligand bound to the receptor, it dimerized and became phosphorylated
- this recruited other multivalent proteins to form a condensate and when the sites became dephosphorylated then the condensate disassembled

Question 5

what are the types of movement proteins can have between compartment? (3)

Answer 5

1. gated movement from the cytosol to the nucleus and back
2. protein translocation or transmembrane transport across membranes such as the ER, mitochondria, plasids, and peroxisomes from the cytosol
3. vesicular transport where vesicles move between compartments and this can go from the cytosol to the ER which can then go to the golgi, endosomes, plasma membrane, peroxisomes, and secretory vesicles

Question 6

what is a signal sequence

Answer 6

a necessary and sufficient amino acid sequence directing a protein to a particular organelle

Question 7

nuclear import signal requirements

Answer 7

positively charged
ex: lys and arg

Question 8

nuclear export signal requirements

Answer 8

hydrophobic and spaced out every 2-3 amino acids
ex: met, leu, phe

Question 9

import to mitochondria signal requirements

Answer 9

alternating positively charged and hydrophobic amino acids
ex:
- hydrophobic: leu, ile, phe, ala
- pos charged: arg, lys

Question 10

import signals into plastids requirements

Answer 10

uncharged and spaced out with a couple of amino acids i between or sometimes just 1
ex: ser, thr

Question 11

import signals into peroxisomes requirements

Answer 11

one uncharged, one positively charged, one hydrophobic
ex:
- uncharged: ser
- pos charged: lys
- hydrophobic: leu

Question 12

import into ER signal requirements

Answer 12

8-10 hydrophobic amino acids
ex: leu, val,gly, ile, phe, trp, ala

Question 13

return to ER signal requirements

Answer 13

one pos charged, 2 neg charged, 1 hydophobic
ex:
- pos: lys
- neg: asp, glu
hydrophobic: leu

Question 14

cotranslational trnasport

Answer 14

proteins are transported into the lumen of an organelle (usually ER) before while it is finishing being synthesized

Question 15

posttranslational transport

Answer 15

a protein is fully made in the cytosol with a sorting signal and is then kept unfolded by chaperones to be brought somewhere else in the cell
- usually nucleus, peroxisomes, mitochondria, and plastids

Question 16

RER functions

Answer 16

protein synthesis and modification

Question 17

SER functions

Answer 17

lipid synthesis, Ca2+ storage, formation of transport vesicles, hormone modification/synthesis, breakdown of toxins

Question 18

what does SRP do

Answer 18

signal recognition particle(s) act as a ribonucleoprotein complex to recognize and bind to the signal sequence as well as a translational pause domain on the ribosome. Then it brings the complex to the ER membrane to funnel the peptide sequence through the bilayer via Sec61

Question 19

what happens when the SRP-ribosome-peptide complex reaches the ER?

Answer 19

SRP is released once it binds to the receptor on the ER membrane, and the hydrophobic peptide sequence is funneled through the translocator protein. The ribosome is right next to the translocator protein, and energy from translation already happening is used to funnel the protein into the lumen

Question 20

ER signal peptidase

Answer 20

this cleaves the hydrophobic ER signal from the rest of the finished protein once in the lumen of the ER

Question 21

Sec61 complex

Answer 21

the ER protein translocator that removes its plug when receiving ER signal sequences and allows the peptides to be pushed into the ER lumen
- when it recognizes a hydrophobic region of a protein meant to stay in the lipid bilayer, it will open laterally and push the protein out from the complex into the lipid bilayer

Question 22

when a protein has a single pass alpha helical portion, which terminus of the protein is in the lumen and which is in the cytosol?

Answer 22

either the N terminus or the C terminus will be on the inner lumen of the ER as long as it is negatively charged. The positively charged side is on the cytosolic side
Note: the N terminus is what comes out of the ribosome first, so if the N terminus is positive, the peptide will be flipped in the translocator protein to push the negative C terminus in

Question 23

what does transmidase do?

Answer 23

it cleaves the signal of a protein with a hydrophobic membrane spanning domain and simultaneously attaches the C terminus of this protein to an amino group on the pre-GPI intermediate. The result is a GPI anchored protein

Question 24

what is N-glycosylation

Answer 24

precursor oligosaccharides are added to asparagine residue (Asn-X-Ser/Thr) in the ER on proteins. There are 3 glucose and many mannose residue sugars on the oligosaccharide

Question 25

what does oligosaccharyl transferase do?

Answer 25

it attaches the oligosaccharide made on dolichol to a protein at the N-glycosylation site

Question 26

where is the precursor oligosaccharide initially assembled on dolichol before being transported to the other membrane side?

Answer 26

on the cytosolic leaflet of the ER membrane and it gets rotated to the lumen leaflet before N-glycosylation

Question 27

what does ER glucosidase do?

Answer 27

it trims the glucose molecules after proteins are bound to calreticulin/calnexin to continue the timing process of protein folding in the ER and allow a properly folded protein to exit

Question 28

what do caltrculin and calnexin do?

Answer 28

these are lectin proteins (Ca2+ dependent) that bind to incompletely folded proteins with 1 glucose residue to prevent misfolded proteins from leaving the ER. These are stuck on the lumen of the ER membrane

Question 29

what does glucysol transferase do?

Answer 29

this recognizes a protein and adds a glucose molecule back on the oligosaccharide tag to be bound to calnexin again if it is misfolded. Otherwise, it allows it to exit the ER

Question 30

how are improperly folded proteins shipped back out of the ER lumen?

Answer 30

by retro transport through a protein translocation complex after lectins will recognize the mannose deprived oligosaccharide on the protein, chaperones will bind to prevent aggregation, and disulfide isomerases will break disulfide bonds to linearize the protein

Question 31

what does mannosidase do?

Answer 31

it slowly trims the mannose residue on the oligosaccharide to mark it for retrotranslocation

Question 32

describe the components of the protein translator complexes

Answer 32

AAA-ATPase pulls the unfolded proteins into the cytosol, E3 ubiquitin ligase attaches polyubiquitin tags to the unfolded proteins on the cytosolic side, N-glycanase removes oligosaccharide chains and these proteins are degraded by a proteasome

Question 33

describe the unfolded protein response and each protein involved

Answer 33

this occurs because too many misfolded proteins triggers a UPR.
1. IRE1 acts to splice an mRNA that will go to the nucleus after being transcribed into a transcription regulatory protein
2. PERK will dimerize when phosphorylated and reduces translation initiation factors for proteins that are supposed to enter the ER
3. ATF6: this gets moved to the Golgi, and the active domain is cleaved into the cytosol to act as a transcription factor in the nucleus and transcribe UPR genes

Question 34

what happens when you add a protease into a solution without a microsome vs one with a RER microsome?

Answer 34

the RER microsome contents will be safe due to the bilayer and protease not being able to get in but the proteins in the solution without a microsome will be cleaved

Question 35

TOM

Answer 35

the translocator protein on the outer mitochondrial membrane, which recognizes positively charged signal sequences on the N terminus

Question 36

TIM

Answer 36

translocator protein on the inner mitochondrial membrane that allows proteins to move into the matrix

Question 37

what 3 ways can proteins be transported into the mitochondrial matrixes and how?

Answer 37

1. membrane potential occurs on the inner mitochondrial membrane where the intermembrane space is positively charged, and the matrix is negatively charged. The protein has a signal sequence that allows it to move through Tim into the matrix space (+ charged). The H+ gradient is what moves the protein down the gradient
2. HSP70 protein on the inner mitochondrial matrix will bind to Tim in the lumen side and pull the protein through using ATP hydrolysis
3. Mia40: this is in the intermembrane space and will recognize SH groups that come through the TOM complex and will form disulfide bonds and oxidize the protein as it moves through the membrane => the resulding protein is oxidized to form sidulfide bonds form cysteine residue in the intermembrane space where it resides

Question 38

describe the nuclear pore complexes (NPCs)

Answer 38

these are made up of membrane ring proteins, scaffold nucleoporin, and channel nucleoporin as well as a nuclear basket on the lumen and cytosolic fibrils on the cytosol side.There are disordered channel nucleoporins in the very middle creating a gel and these have FG repeats on them for binding

Question 39

nuclear import receptors (importing)

Answer 39

proteins that recognize NLS cargo proteins (bind) which then bind to FG repeats to locally disband the gel and let the protein cargo in

Question 40

describe how GAP and GEF work with Ran for nuclear import

Answer 40

these are kinases that work with GTPase proteins like Ran to mobilize hydrolysis. when Ran-GDP is in the cytosol it gets transported to the nuclear side and interacts with GEF which replaces the GDP for GTP. This Ran-GTP now binds with a cargo protein site to kick out the cargo and attach to the receptor protein before being taken back out of the nucleus. On the outside, GAP interacts with Ran-GTP to hydrolyze it and once again turn in back into Ran-GDP as it dissociates from the receptor protein. This cycle repeats.

Question 41

describe nuclear export

Answer 41

This is the opposite of nuclear import where a cargo protein will bind with the receptor protein as well as Ran-GTP (promotes it) as it is exported out of the nucleus. On the outside the cargo is released as GAP hydrolyzes Ran-GTP to GDP. The receptor protein then diffuses back into the nucleus alongside Ran-GDP to restart the cycle.