L5 - Atg8 Conj Sys

Question 1

what was the first experiment in the Atg8 conjugation system?

Answer 1

1) made cells Δatg8 N-term myc-Atg8 and Δatg8 C-term myc-Atg8
RESULT: observed that only N-term myc-Atg8 was observable when using anti-myc Ab, indicating that C-term was cleaved off somehow

Question 2

which protein was found to cleave the C-term of Atg8?

Answer 2

Atg4

Question 3

how did they figure out that Atg4 was the protein that cleaved off the C-term of Atg8?

Answer 3

1) ran separate WB experiments with single deletions of each Atg protein and C-term myc-Atg8
2) observed for which mutant resulted in loss of myc-Atg8 band
RESULT: found that in the Atg4 mutant, there was no band for myc-Atg8

Question 4

what kind of protein is Atg4?

Answer 4

protease

Question 5

what are the 4 major classes of proteases?

Answer 5

1) Ser protease
2) Cys protease
3) Asp protease
4) metalloprotease

Question 6

how did they figure out what kind of protease Atg4 was?

Answer 6

1) mixed together myc-Atg8 and Atg8 in a WT cell lysate (that has Atg4 too)
2) added inhibitors that are specific for each type of protease
3) used anti-Atg8 Ab
4) observed which lane will have no C-term myc-tagged Atg8 cleavage

Question 7

Cys protease inhibitor

Answer 7

NEM

Question 8

Ser protease inhibitor

Answer 8

PMSF

Question 9

Asp protease inhibitor

Answer 9

pepstatin

Question 10

metalloprotease inhibitor

Answer 10

1,10-phenanthroline

Question 11

what kind of protease is Atg4?

Answer 11

cys protease

Question 12

where exactly does Atg4 cleave Atg8?

Answer 12

second last Gly residue on Atg8

Question 13

how did they prove that Atg4 cleaved the second last Gly residue of Atg8?

Answer 13

1) expressed C-term myc-Atg8 and variants with second last residue (Gly) deleted or replaced)
2) did WB and added anti-Atg8 AB)
3) variants with last G replaced/substituted resulted in no cleaved Atg8 band
RESULT: Atg4 cleaves at second last AA residue on Atg8 (Gly)

Question 14

what is Atg8 conjugated to?

Answer 14

free amine group of PE (major membrane lipid)

Question 15

why was the Atg8 band higher than the Atg8-PE band?

Answer 15

different kind of gel system, not really by weight

Question 16

what proteins other than Atg4 is required for the Atg8 conjugation system?

Answer 16

Atg7 and Atg3

Question 17

how did they figure out if Atg7 binds to Atg8?

Answer 17

co-immunoprecipitation

Question 18

how does co-immunoprecipitation work?

Answer 18

1) cell lysate
2) add Ab specific to a protein of interest
3) add protein (that’s specific to the Ab) conjugated bead
4) protein bead binds to Ab
5) wash
6) WB analysis to figure out which proteins were bound to the Ab (will be more than one if a complex forms)

Question 19

was the sequence of Atg8 similar to Atg12?

Answer 19

yes

Question 20

what was the result of the co-IP experiment with Atg7 and Atg8?

Answer 20

Atg7 interacts with Atg8

Question 21

what is required for Atg8 to bind to Atg7?

Answer 21

C-terminal glycine of Atg8 needed to interact with Atg7

Question 22

what was the E2 enzyme in the Atg8 conjugation system?

Answer 22

Atg3

Question 23

how did they figure out that Atg3 was the E2 enzyme in the Atg8 conjugation system?

Answer 23

did not go over, just know

Question 24

what is the E3 enzyme in the Atg8 conjugation system?

Answer 24

Atg12-Atg5 conjugate (from the Atg12 conjugation system) promotes the formation of the Atg8-pE

Question 25

what was cool about the relationship between Atg8, Atg12, and ubiquitin?

Answer 25

X-ray crystallography showed that Atg8 and Atg12 adopted very similar 3D structures of ubiquitin despite lacking homology at the AA sequence level

Question 26

what determines the substrate specificity in the Ubiquitin system?

Answer 26

E3 enzyme

Question 27

what determines the substrate specific in the Atg8 conjugation system?

Answer 27

Atg3 (the E2-like enzyme, not the E3 enzyme like in ubiquitin)

Question 28

what are the similarities between ubiquitination, Atg12 and Atg8 conjugation system?

Answer 28

1) ATP-dependent activation step
2) E1 and E2 reactions involve formation of thioester bond
3) C-term glycine of ubiquitin, Atg12 and Atg8 is involved in the final conjugation

Question 29

what are the differences between ubiquitination, Atg12 and Atg8 conjugation system?

Answer 29

1) only one copy of Atg12 and Atg8 ends up being bound to its target
2) conjugated products from the Atg12 and Atg8 systems are not degraded
3) Atg8 requires initial proteolytic step first (Atg4 cleaves C-term)
4) Atg8 conjugated to lipid instead of protein)