L4 - Atg1 & Atg12 Conj Sys

Question 1

how did they really prove that Atg13 was REALLY phosphorylated

Answer 1

grew cells in 32P-containing media and performed IP

• half of the cells were treated w phosphatase
• saw cells with phosphatase treatment had reduced smearing so the phosphatase actually did work

Question 2

how did they find the phosphorylation sites for Atg13?

Answer 2

knew there were 8 potential spots by mass spec and based on flanking sequences

• made Atg13-8SA where the Ser->Ala so no phosphorylation can occur
• put into ΔAtg13
• tight bands for Atg13-8SA (no phosphorylation)

Question 3

where is Atg13 phosphorylated?

Answer 3

8 different ser residues

Question 4

how did they show that TOR phosphorylates Atg13?

Answer 4

1) purify TORC1 from yeast (encodes FLAG-KOG1 that is a part of TORC1 and HA-TOR1)
2) did in vitro kinase assay with TF-tagged Atg13 as substrate
3) autoradiography to detect phosphorylation
4) showed that Atg13 is phosphorylated by TORC1 and removing the phosphorylation sites basically removes this

Question 5

how did they measure the amount of Atg13 used in the reaction?

Answer 5

CBB (Coomassie blue staining)

Question 6

what was the substrate in this assay?

Answer 6

TF-tagged WT Atg13 or Atg13-8SA

Question 7

where is Atg 12 relative to the Atg hierarchy?

Answer 7

late stages of autophagy

Question 8

did Atg12 mutants result in AB accumulation upon nitrogen starvation?

Answer 8

no, only in the WT vacuole

Question 9

what was the conclusion made when Atg12 mutants were shown to not have AB accumulation upon starvation?

Answer 9

Atg12 essential for autophagy

Question 10

what was unusual when Noboru transformed Δatg12 yeast strains with HA-Atg12, prepared lysates and performed WB with HA-Ab?

Answer 10

there were two equal size bands that formed, one of predicted mass and one that was much heavier

Question 11

what was the conclusion drawn from the appearance of a higher molecular band appearing in the Wb of Atg12?

Answer 11

Atg12 might be covalently linked to something (other protein)

Question 12

how did Noboru figure out which proteins were bound to Atg12?

Answer 12

prepared cell lysates with HA-tagged Atg12 and Atg gene mutations/deletions of other Atg proteins

• Wb
• observed for loss of double band

Question 13

which proteins were found to bind to Atg12?

Answer 13

Atg5, Atg7 and Atg 10

Question 14

how do we know that the Atg12 complex is formed via covalent interactions?

Answer 14

Wb treatment removes non-covalent interactions

Question 15

how do we know that Atg12 binds to Atg5 directly?

Answer 15

Wb with HA-Atg5 and anti-HA Ab. Two bands form in WT but only one band forms in Δatg12

Question 16

what is required for Atg12 to bind to Atg5?

Answer 16

Atg12 C-term Gly

Question 17

how did Noboru prove that the C-term Gly is required for Atg12 to bind to Atg5?

Answer 17

Wb with anti-HA Ab and WT HA-Atg12 and HA-Atg12 mutants with no C-term Gly. Only the WT-Atg12 with the C-term Gly was able to develop a band corresponding to the Atg12 complex

Question 18

how did they prove that the Atg5 conjugation to Atg12 is required for autophagy?

Answer 18

ALP activity assay where they deleted/mutated the C-term Gly of Atg5. No upregulation of autophagy upon starvation

Question 19

How is Atg12 similar to ubiquitination

Answer 19

Atg5’s C-term Gly covalently linking to Atg12 similar to how ubiquitins C-term Gly is covalently linked to Lys of substrate?

Question 20

How did Noboru show that Atg12 linking to Atg5 was similar to ubiquitin where the C-term Gly links to a Lys?

Answer 20

Mutate each Lys to R in Atg 5

• express mutant Atg 5’s in Δatg5 strain
• prepare cell lysate and Wb using anti-HA Ab
• see which mutant does not have heavy band corresponding to Atg5 and Atg12 complex
• K149R mutant had no heavy band

Question 21

how does Atg12 bind to Atg5?

Answer 21

Atg12 C-term Gly covalently links to K149 of Atg5 in similar fashion to ubiquitin

Question 22

what was the first hint that Atg7 acted as an E1 enzyme in the Atg12/5 conjugation system?

Answer 22

Atg7 had sequence homology to Uba1 (an E1 enzyme)

Question 23

what is the distinguishing factor of E1 in ubiquitin pathway?

Answer 23

E1 requires ATP

Question 24

how did they prove that the Atg7 was the E1 enzyme in the Atg12 conjugation system

Answer 24

1) prepared Δatg5 w HA-Atg12 and Δatg12 w HA-Atg5
2) mixed the cell lysates
3) add ATP to one tube and no ATP in another tube
4) observe whether the conjugated product formed
RESULT: only the ATP tube had conjugated HA product

Question 25

Did Atg10 had similar sequence to any E2 proteins?

Answer 25

No

Question 26

How did they prove that Atg10 was the E2 enzyme?

Answer 26

Won’t go into it, they just figured it out

Question 27

How does Atg10 bind to Atg12?

Answer 27

thioester bond between Cys of Atg10 to Atg 12 (like how it would happen in E2 reactions)

Question 28

How did they figure out which Cys residue in Atg10 binds to Atg12?

Answer 28

1) use separate plasmids to generate HA-Atg12 and HA-Atg10 (WT and Cys->Ser mutants in three positions)
2) prepare cell lysates and use anti-HA ab
RESULT: found that mutation in Cys133 of Atg10 resulted in no heavy band containing Atg12 bound to Atg5

Question 29

What is the residue in Atg10 that is important for the Atg12 conjugation system?

Answer 29

Cys133

Question 30

What was the E3 enzyme in the Atg12 conjugation system?

Answer 30

Atg10 acts as the E3 enzyme as well to transfer Atg12 to Atg5

Question 31

why is the Atg12 conjugation system important?

Answer 31

plays important role in second ubiquitin-like system involving Atg8