Secretion: First, Second, Third

Question 1

Are proteins post or cotranslationally inserted into the ER from the ribosome?

Answer 1

Cotranslationally

Question 2

How was secretion initially researched?

Answer 2

Secretion was studied with pancreatic acinar cells using synthesized digestive enzymes (which are trafficked to the digestive tract). The cells have membrane-bounded compartments filled with zynogen (digestive enzyme) granules.

Question 3

Describe the pulse-chase technique.

Answer 3

Take a cell, add radioactivity to a biological molecule, and follow the radioactivity as it moves through the cell.
Radioactive leucine was added, then chased with nonradioactive leucine. Over time, they watched where the radioactive wave went.

Question 4

Why was radioactive leucine selected for the pulse chase technique?

Answer 4

Leucine is an essential amino acid the cell cannot synthesize; must be taken up from the environment

Question 5

Why were pancreatic acinar cells used for the pulse-chase experiment?

Answer 5

Palade wanted to study cells that secreted lots of proteins. The pancreatic acinar cells secreted digestive enzymes called zynogens to the digestive tract.

Question 6

What path do the granules in pancreatic acinar cells travel?

Answer 6

They are released in the pancreas’s duct, then move into the intestines.

Question 7

What was used originally for the pulse-chase technique? What is used now?

Answer 7

Originally, Palade used radioisotopes (radioactive leucine). Now, fluorescent and photo-activated molecules are used more often.

Question 8

What does the color red indicate in a pulse-chase technique?

Answer 8

Indicates radioactivity is present in this organelle - hot

Question 9

What does the color red indicate in a pulse-chase technique?

Answer 9

Indicates radioactivity is not present in this organelle - cold

Question 10

What would happen if the pulse was not chased in the pulse-chase technique?

Answer 10

The radioactivity would eventually light up all the organelles, making it impossible to trace a secreted protein’s path.

Question 11

What are the two versions of the pulse-chase technique?

Answer 11

EM radiography
Cell fractionation

Question 12

How was the pulse-chase experiment’s results viewed in an electron micrograph?

Answer 12

Radiation causes silver ions to precipitate in the organelle with irradiated proteins; they appear as black spots. Micrographs were created at different times to track the progression of the radiation.

Question 13

What happens when digestive proteins from the pancreatic acinar cells are secreted into the pancreatic duct?

Answer 13

Nitrogen granules begin digesting.

Question 14

What didn’t pulse-chase reveal?

Answer 14

Palade was unsure if the secreted proteins were near the organelles or if they were associated with them, especially the ER.

Question 15

What did pancreatic cell fractionation reveal to Palade?

Answer 15

The pulse-chase experiment was accurate. The fractionation of purified rough ER indicated the secreted proteins were associated, not just near, the rough ER.

Question 16

How did pancreatic cell fractionation work?

Answer 16

Radioactivity was added to pancreatic cells via the pulse-chase technique. Samples were taken and fractionated at different times and each fraction was examined for radioactivity (based on location of marker organelles).

Question 17

What did using GFP-Labeled VSV Proteins do?

Answer 17

Allowed cultured mammalian cells to be used to discover more about the secretory pathway in living cells; was able to see a video of a protein migrating through the pathway in real time

Question 18

Describe the GFP fusion protein.

Answer 18

Temperature sensitive - at normal temperatures, the mutant appears to be wild type, but by raising 1-2 degrees Celsius, the protein denatures and loses the ability to move through the secretory pathway.

Question 19

What happens if a GFP fusion protein is returned to a normal temperature?

Answer 19

It may regain its normal conformation and move through the pathway.

Question 20

How was the mechanism for the secretory pathway discovered?

Answer 20

By using temperature sensitive yeast secretory mutants. Creating mutants via chemical mutagens, then searching for conditional mutants with defects in the secretory pathway. Identifying the genes responsible for the secretory pathway enabled the finding of the proteins and eventually the mechanism.

Question 21

What are conditional mutants?

Answer 21

mutants where the effect of the mutation was only visible at high temperatures

Question 22

How were mutants with defects in the secretory pathway identified?

Answer 22

Via centrifugation. If the secretory pathway were inoperable, proteins continue being made that cannot exit the cell. Constipated cells are denser.

Question 23

How does double mutant analysis work?

Answer 23

Take 2 mutant strains, assign classes based on phenotype, then mate together 2 different classes and see which parent the offspring most resembles. Whichever is most resembled, the organelle the mutation is in comes first in the pathway. Using different combinations helps one figure out the sequence within the pathway.

Question 24

What is a Class B mutant?

Answer 24

Mutant with an accumulation in the rough ER; problem with budding of vesicles from the Rough ER

Question 25

What is a Class D mutant?

Answer 25

mutant with an accumulation in the Golgi; problem with transporting from the Golgi to the secretory vesicles

Question 26

What is a Class E mutant?

Answer 26

mutant with an accumulation in the secretory vesicles; problem with transport from secretory vesicles to the cell surface