Lecture 3

Question 1

What are the 18 Atg?

Answer 1

Core autophagy proteins.
Initiation of autophagy, generation of autophagosomes,
localization to PAS upon Nitrogen starvation.
17 = critical upstream factor

Question 2

What does Atg1 depend on to localize to PAS?

Answer 2

atg1, atg13

Question 3

What are 5 functional groups of Atg proteins?

Answer 3

1. Atg1 kinase and its regulators
2. Autophagy - specific PI 3 kinase complex
3. Atg12 conjugation system
4. Atg8 conjugation system
5. Atg2-Atg18 complex and Atg9

Question 4

Describe the alkaline phosphatase (ALP) assay

Answer 4

Measures amount of pho8^60 delivered to vacuole.
Determined activity of phosphate which correlates with level of autophagy within living cell (yeast).
First quantitative autophagy, showing that Atg1 is essential.

Question 5

Is kinase activity of atg1 required for autophagy?

Answer 5

Yes. K54A mutant has low AP activity.

Question 6

Describe Rapamycin

Answer 6

Immunosuppressive which inhibits TOR. It is a Ser Thr protein kinase of 2500 residues, which regulates cell growth. Also a Phosphatidyinositol kinase homologue which conrols autophagy in yeast, Atg1 is upregulated by Rapa treatment.

Question 7

Describe role of TOR in autophagy

Answer 7

TOR is inhibited by rapamycin. TOR artificially regulates autophagy (convinces body is in starvation, even in nutrient-rich environment.) Autophagy activated when TOR activity is downgraded. TOR is needed to mediate autophagy pathway. TOR1 is not essential for viability, while TOR2 is. These protein isoforms can form TORC1, TORC2.