Section 6C: Protein sorting into the Mitochondria and ER

Question 1

Any protein that wants to end up in any organelle other than the Nucleus or the Mitochondria gets sorted into the…

Answer 1

Endoplasmic Reticulum

Question 2

Transport from the Cytosol to the ER because…

Answer 2

ER is the first place of transit for the subsequent movement of most proteins

Question 3

The Endoplasmic Reticulum (ER)

Answer 3

1. Membrane labyrinth of tubules, sheets and sacs: ~50% of total surface membrane and 10% of cell volume
2. Lipid Synthesis: where phospholipids and cholesterol get made
3. Protein Synthesis
4. Lipid and proteins for ER, Golgi, plasma membrane, endosomes, lysosomes and extracellular space
5. Ca+2 Storage
6. Specialized domains: rough ER, smooth ER and sarcoplasmic reticulum

Question 4

The rough ER

Answer 4

• coated with ribosomes (little dots on surface of the ER)
• proteins are made by ribosomes
• active area of protein synthesis

Question 5

Post-translational sorting

Answer 5

• Sorting happens after translation or protein synthesis is complete
• First, make the entire protein, have it adopt to its 3D structure, and the protein gets sorted into the right compartment

Question 6

Co-translational sorting

Answer 6

• Protein is not finished and gets sorted while they’re being made by ribosome
• Still in the process of being polymerized

Question 7

Why do proteins who go to the ER have to sort co-translationally instead of being made first?

Answer 7

• a lot of these proteins will end up being integral membrane proteins that don’t necessarily go to the interior of the ER, but go to the membrane of the ER
• Because integral membrane proteins have hydrophobic non-polar pieces, they need to be inserted into the membrane as they are being made
• if it was sorted post-translationally, you’d be expecting the piece of the protein that’s hydrophobic to live in a hydrophilic or water-based cytosol

Question 8

An integral membrane protein starts its life by…

Answer 8

getting sorted into the ER

Question 9

If a protein is sorting to the endoplasmic reticulum, are we dealing with Co-translational sorting or Post-translational sorting?

Answer 9

Co-translational sorting because a lot of the proteins, that sort to the ER have hydrophobic amino acids

Question 10

Why do these proteins have hydrophobic amino acids?

Answer 10

• Because they have to pass through the lipid bilayer
• many of them are integral membrane proteins that have hydrophobic regions that can sit in the hydrophobic part of the lipid bilayer
• those parts are not very useful in the cytosol thus they have to be sorted as they’re being made otherwise the protein is too unstable

Question 11

How are proteins targeted to the ER?

Answer 11

1. Signal sequence: an address that tells the cell where the protein has to go; for sorting to the ER
2. Signal recognition particle (SRP): signal sequence is then going to be recognized by SRP
3. SRP receptor
4. Translocation by translocator channel

Question 12

What is the first thing that is encountered as the protein emerges out of the ribosome?

Answer 12

The signal sequence

Question 13

What is the goal of the signal sequence?

Answer 13

to be able to target the ribosome so that it now locates and starts inserting the protein in the endoplasmic reticulum as the protein is being made.

Question 14

What does the Signal Recognition Particle (SRP) recognize?

Answer 14

the ER signal peptide sequence

Question 15

What is the SRP made out of?

Answer 15

It is a molecule hat is made up a part of RNA and a part of protein

Question 16

What does the SRP first bind to?

Answer 16

• binds to any ribosome that is making a protein where the first part of it is the signal sequence
• so if a ribosome is synthesizing a protein and that protein has a signal sequence to sort to the ER, then it will combine it

Question 17

What is the point of binding SRP to ribosome?

Answer 17

help steer the ribosome to the surface of the ER

Question 18

What else does SRP do?

Answer 18

• it causes translation to pause because protein could be made and then could be damaged since it has to stay in the cytosol
• recognize and bind to ER signal peptide
• direct this all to the ER translocation pore

Question 19

What is the purpose of the translation pause?

Answer 19

• keeps the ribosome from making the rest of the protein until it’s actually gone to the ER
• prevents the ribosome from accidentally completing the process of translation
  before the cells had a chance to start sorting it into the ER

Question 20

How does the SRP go to the ER?

Answer 20

• it binds to the SRP Receptor
• the SRP binds to the Ribosome and eventually connect with something on the ER called the SRP Receptor

Question 21

What is the SRP receptor?

Answer 21

it is an integral membrane protein with a transmembrane domain in the ER (it is anchored into the ER)

Question 22

What is the whole point if the SRP receptor?

Answer 22

to bring the ribosome to the pore

Question 23

How is translocation accomplished?

Answer 23

• core of translocator is the Sec61 complex: three polypeptide chains
• translocator forms aqueous pore: pore can’t be open all the time; only opens when protein comes in and immediately closes after otherwise interior of ER would leak out
• pore is gated to maintain ER membrane impermeability
• pore opens when nascent polypeptide chain enters

Question 24

The Sec61 protein translocator has 2 states

Answer 24

1. Open:
   - when a protein binds to the pore, the plug is removed, making space for the nascent protein (being synthesized) to pass through
   - translocator pore opens wide enough for signal sequence to be wedged into it, and the rest of the protein threads though so that translation is done
2. Closed:
   - Sec61 has a plug that blocks the pore when there are no proteins to be transported

Question 25

Proteins have ends with different chemical groups

Answer 25

Amino terminus (N-terminus) and Carboxylic terminus (C-terminus)

Question 26

Which end do proteins get made from?

Answer 26

Proteins always get made from the N-terminus to the C-terminus

Question 27

What end of the protein is the signal sequence?

Answer 27

N-terminus because it is the first one that gets made during protein synthesis and amino acids get added on from here until the protein is complete; thus the first thing that is going to emerge from the ribosome is this part of the protein

Question 28

Three categories of proteins inserted into the Sec61 pore

Answer 28

1. Translocation of proteins with a terminal signal sequence: signal sequences that are there from beginning to end 2. Translocation of proteins with an internal signal sequence 3. Translocation of proteins with a multiple signal sequence (and stop transfer sequences)

Question 29

Category 1: Translocation of proteins with a terminal signal sequence

Answer 29

- one type of protein that have this is an integral membrane protein 1. Signal sequence is recognized: opens Sec61 pore, signal sequence wedged in pore, rest of the protein translocates through 2. Stop transfer sequence is recognized: exits though the side of the Sec61 pore, becomes transmembrane domain (hydrophobic, non polar), translocator pore waits for the hydrophobic stretch of amino acids which allows protein to escape to the hydrophobic core of lipid bilayer 3. Signal sequence is cleaved

Question 30

Signal peptidase

Answer 30

cuts off original signal sequence however, the transmembrane domain does not get cut off that stays in the lipid bilayer

Question 31

What happens if there is no stop transfer sequence?

Answer 31

Whole protein gets synthesized and all of it ends up in the ER which eventually gets secreted or released from the cell (e.g. insulin)

Question 32

Summary Category 1: Translocation of proteins with a terminal signal sequence

Answer 32

•N-terminal signal sequence binds and opens pore of translocator; signal sequence = start-transfer signal •Signal sequence contacts translocator wall and lipid bilayer •Pore opens laterally (seam) to allow diffusion of signal sequence, which remains embedded in membrane • Does the protein also have a stop-transfer sequence? Yes - this becomes a transmembrane domain; protein is a transmembrane protein No - this becomes a soluble protein in the lumen of the ER - secreted protein •Signal peptidase cleaves off the (terminal) signal sequence

Question 33

Not all proteins have a terminal (end) signal sequence

Answer 33

- Some proteins have a signal sequence in the middle of the protein thus, it doesn't get sorted into the ER right away - Internal signal sequence cannot be cut off since it is in the middle

Question 34

Signal sequence in the middle of the protein is...

Answer 34

a transmembrane domain

Question 35

Category 2: Translocation of proteins with an integral signal sequence

Answer 35

- proteins with an integral signal sequence are inserted into the Sec61 pore to allow the rest of the protein to translocate through the pore - Directions of insertion by charges of amino acids around the internal signal sequence - Internal signal sequence is NOT cleaved off

Question 36

Category 3: Translocation of proteins with a multiple signal sequence (and stop transfer sequences)

Answer 36

- Multipass transmembrane proteins are made by multiple repeats of: 1. Integral signal sequence 2. Stop transfer sequence

Question 37

How do multipass transmembrane proteins get sorted?

Answer 37

They have multiple start transfer and stop transfer sequences

Question 38

What happens to proteins after insertion into the ER membrane or lumen?

Answer 38

- Protein reporter: VSVG-fused to GFP - Protein starts off in ER - By about t = 50 mins the protein is mostly in the Golgi - By about t = 1h 30 min the protein begins to accumulate at the plasma membrane