L5 - Atg8 Conj Sys Flashcards
what was the first experiment in the Atg8 conjugation system?
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
which protein was found to cleave the C-term of Atg8?
Atg4
how did they figure out that Atg4 was the protein that cleaved off the C-term of Atg8?
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
what kind of protein is Atg4?
protease
what are the 4 major classes of proteases?
1) Ser protease
2) Cys protease
3) Asp protease
4) metalloprotease
how did they figure out what kind of protease Atg4 was?
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
Cys protease inhibitor
NEM
Ser protease inhibitor
PMSF
Asp protease inhibitor
pepstatin
metalloprotease inhibitor
1,10-phenanthroline
what kind of protease is Atg4?
cys protease
where exactly does Atg4 cleave Atg8?
second last Gly residue on Atg8
how did they prove that Atg4 cleaved the second last Gly residue of Atg8?
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)
what is Atg8 conjugated to?
free amine group of PE (major membrane lipid)
why was the Atg8 band higher than the Atg8-PE band?
different kind of gel system, not really by weight
what proteins other than Atg4 is required for the Atg8 conjugation system?
Atg7 and Atg3
how did they figure out if Atg7 binds to Atg8?
co-immunoprecipitation
how does co-immunoprecipitation work?
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)
was the sequence of Atg8 similar to Atg12?
yes
what was the result of the co-IP experiment with Atg7 and Atg8?
Atg7 interacts with Atg8
what is required for Atg8 to bind to Atg7?
C-terminal glycine of Atg8 needed to interact with Atg7
what was the E2 enzyme in the Atg8 conjugation system?
Atg3
how did they figure out that Atg3 was the E2 enzyme in the Atg8 conjugation system?
did not go over, just know
what is the E3 enzyme in the Atg8 conjugation system?
Atg12-Atg5 conjugate (from the Atg12 conjugation system) promotes the formation of the Atg8-pE
what was cool about the relationship between Atg8, Atg12, and ubiquitin?
X-ray crystallography showed that Atg8 and Atg12 adopted very similar 3D structures of ubiquitin despite lacking homology at the AA sequence level
what determines the substrate specificity in the Ubiquitin system?
E3 enzyme
what determines the substrate specific in the Atg8 conjugation system?
Atg3 (the E2-like enzyme, not the E3 enzyme like in ubiquitin)
what are the similarities between ubiquitination, Atg12 and Atg8 conjugation system?
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
what are the differences between ubiquitination, Atg12 and Atg8 conjugation system?
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)
