Protein Targeting to the ER

Question 1

For what final destinations are proteins required to go through the ER?

Answer 1

ER, Golgi, lysosomes, plasma membrane, outside the cell

Question 2

What 3 things are common to all proteins that go to the ER?

Answer 2

1. Translation and import happen simultaneously
2. Modifications to the proteins occur in the ER lumen
3. Once inside the secretory pathway, are transported in vesicles

Question 3

How did we find out that secretory proteins were in the ER?

Answer 3

When homogenized, the ER breaks up into microsomes. When detergent was added to the homogenate, the labeled secretory proteins got broken down by proteases that were added. Without the detergent, the labeled proteins were safe from the proteases inside the microsomes

Question 4

How did we find out that translation occurred during import to the ER?

Answer 4

Secretory proteins were created in vitro and then microsomes were added, and the proteins didn’t go in. When the microsomes were there in the first place, the proteins were in the ER

Question 5

What is it called when translation and import occur simultaneously?

Answer 5

Co-translational import

Question 6

How is co-translational import different from how nucleus proteins are translated and imported?

Answer 6

Nucleus transport is post-translational import. The entire protein is made and then it is imported, it’s a two step process. Transport to the ER has both translation and import occurring in the same single step

Question 7

What are the characteristics of the ER signal sequence?

Answer 7

Is the first 16-30 amino acids for soluble proteins and some membrane proteins. Has a few positively charged amino acids next to a stretch of hydrophobic amino acids

Question 8

How is the ER signal sequence different from an NLS in terms of what happens to it once it has done its job?

Answer 8

It gets cleaved off. The NLS usually stays and gets folded into the proteins as per usual

Question 9

What are the steps to synthesize a soluble ER protein?

Answer 9

1. The ribosome translated the N-terminus ER signal sequence
2. SRP recognizes the ER signal sequence and binds to it
3. Translation is paused
4. SRP brings the ribosome, the mRNA, the polypeptide chain and all the translation helpers to the ER membrane
5. SRP binds to the SRP receptor and they both hydrolyze their GTP to GDP
6. The translocon opens and translation resumes
7. The protein gets threaded through the translocon into the ER and signal peptidases cleave off the signal sequence
8. Translation is completed, the ribosome dissociates and the translocon closes

Question 10

What key event occurs during translation into the ER to get a membrane protein?

Answer 10

A particularly hydrophobic stretch of amino acids gets stuck in the translocon, and causes the rest of the protein to be translated in the cytoplasm

Question 11

Once a membrane protein is made, does its orientation stay constant when it is transported to other locations in the endomembrane system? (yes/no)

Answer 11

Yes

Question 12

What are the steps to make a type 1 membrane protein (single pass, N-terminus in the ER lumen)?

Answer 12

1. Co-translational import as per usual
2. The stop transfer anchor sequence gets translated
3. The stop transfer anchor sequence binds to the translocon
4. The rest of the protein is synthesized on the cytoplasmic side of the ER membrane
5. Translation is completed and the protein diffuses laterally into the membrane

Question 13

What are the characteristics of the stop-transfer anchor sequence? What does it become in the completed protein?

Answer 13

Has a lot of hydrophobic amino acids, it becomes the membrane spanning domain

Question 14

What are the steps to make a type 2 membrane protein (single pass, N-terminus in the cytoplasm)?

Answer 14

1. The ribosome starts translating the protein, but some of the protein gets translated before the signal sequence shows up
2. SRP binds to the signal sequence in the middle of the protein and transports the ribosome + friends to the ER membrane
3. The signal sequence is also the anchor sequence, and it gets inserted into the translocon to form the transmembrane domai
4. The rest of the protein get translated into the ER lumen
5. The protein diffuses laterally into the membrane and the translocon closes
   Note: can also be vice versa, with the stop transfer anchor sequence first

Question 15

What are the steps to make a multiples proteins that goes through the membrane twice?

Answer 15

1. The protein gets translated until it hits a start transfer signal anchor sequence
2. Ribosome and friends get translocated to the ER membrane by SRP
3. The start transfer signal anchor sequence gets put into the translocon and the protein continues to get translated into the ER lumen
4. The stop transfer anchor sequence gets translated and gets stuck in the translocon
5. The remainder of the protein gets translated in the cytoplasm

Question 16

How do type 1 and type 2 membrane proteins differ?

Answer 16

Type 1 has the N-terminus in the ER lumen and has 2 hydrophobic regions: the ER signal sequence and the stop transfer anchor sequence
Type 2 has the N-terminus in the cytoplasm and has one hydrophobic region: the start transfer signal anchor sequence