Lecture 8

Question 1

Why must proteins that go into the mitochondria be in an unfolded state?

Answer 1

• Makes matrix-target sequence more easily accessible

- Easier to go through 2 membranes (translocons)

Question 2

Where is the matrix-target sequence located on a protein?

Answer 2

N-terminus

Question 3

Describe the process of protein import into the mitochondrial matrix.

Answer 3

1. Protein is unfolded by cytosolic Hsc70
2. Matrix-target sequence binds to import receptor on outer membrane of cell
3. Protein is transported through translocon of outer membrane
4. Protein is transported through translocon of inner membrane
5. Matrix Hsc70 keeps protein unfolded as it enters mitochondrial matrix
6. Matrix processing protease cleaves targeting sequence
7. Protein folds into its active form

Question 4

What does the “end” of the protein refer to? “Beginning”?

Answer 4

• End = C-term

- Beginning = N-term

Question 5

If you fuse mitochondrial matrix targeting signal sequence to the end of GFP, where would you expect to find GFP? Explain.

Answer 5

Cytoplasm b/c matrix targeting signal is on the C-term, but must be on N-term to get to the mitochondria

Question 6

If you put mitochondrial matrix targeting signal sequence in the middle of GFP, where would you expect to find GFP? Explain.

Answer 6

Cytoplasm b/c matrix targeting signal must be on the N-term

Question 7

Describe the process of protein import into the inner mitochondrial membrane.

Answer 7

1. Matrix-targeting sequence binds to import receptor on outer membrane
2. Protein passes through translocon on outer membrane
3. Protein passes through protein on inner membrane
4. Matrix-targeting sequence is cleaved
5. Stop-transfer sequence prevents further transport of protein –> part of protein is in the inner membrane, the other part is in the intermembrane space

Question 8

What makes the proteins targeting the inner mitochondrial membrane different than proteins going to the mitochondrial matrix?

Answer 8

Proteins going to the inner mitochondrial membrane have a stop-transfer sequence in the middle

Question 9

Explain the pulse-chase experiment in pancreatic exocrine cells as conducted by Palade (1960s).

Answer 9

1. Cells incubated in [H3] - leucine solution
2. Wash off label
   3a. At 0 mins, precipitate is on ER
   3b. At 7 mins, precipitate is on golgi
   3c. At 37 mins, precipitate is on condensing granules
   3d. At 117 mins, precipitate is on the lumen

Question 10

If proteins accumulate in the cytosol, what function is defective?

Answer 10

Transport into the ER

Question 11

If proteins accumulate in the rough ER, what function is defective?

Answer 11

Budding of vesicles from the ER

Question 12

If proteins accumulate in the ER-to-Golgi, what function is defective?

Answer 12

Fusion of transport vesicles w/ Golgi

Question 13

If proteins accumulate in the Golgi, what function is defective?

Answer 13

Transport from Golgi to secretory vesicles

Question 14

If proteins accumulate in the secretory vesicles, what function is defective?

Answer 14

Transport from secretory vesicles to cell surface

Question 15

A sec12 mutant yeast cell has a lack of vesicles and a larger ER as compared to wild-type. A sec17 mutant has an excess of vesicles and a small ER as compared to wild-type. What would you predict a sec12, sec17 double mutant would look like?

Answer 15

Too few vesicles and a large ER b/c first mutant (sec12) dominates

Question 16

What are sec mutants?

Answer 16

Mutations of yeast genes that disrupt steps of the secretory pathway

Question 17

What are the 2 directions proteins can move through the secretory system?

Answer 17

• Anterograde

- Retrograde

Question 18

What is anterograde trafficking?

Answer 18

Forward trafficking of proteins from the ER –> Golgi –> plasma membrane

Question 19

What is retrograde trafficking? Significance?

Answer 19

Backward movement of proteins from the plasma membrane –> Golgi –> ER
**Endocytosis to replenish membranes back to organelles

Question 20

What are the 2 default locations for any protein made in the ER?

Answer 20

• Secreted out of the cell

- Plasma membrane

Question 21

Where do soluble proteins made and inserted into the ER lumen go by default?

Answer 21

Secreted out of the cell

Question 22

Where do transmembrane proteins go by default?

Answer 22

Plasma membrane

Question 23

What are the 3 major steps of protein secretion (membrane targeting)?

Answer 23

1. Protein is synthesized on the cytoplasmic surface of the ER and translocated into the ER lumen
2. Proteins modified w/ sugars as they travel through from ER to Golgi to plasma membrane
3. Proteins packaged into vesicles for transport from compartment to compartment.
   - vesicles bud from one compartment, move to another, and fuse there.