The document summarizes research on Atg15, a factor involved in autophagy and lipophagy in Saccharomyces cerevisiae. It finds that atg15Δ strains have a decreased number of lipid bodies and BODIPY staining diffuses in the vacuole, suggesting Atg15 is important for normal lipid body formation and degradation. An Erg6-GFP processing assay is proposed to further study Atg15's role in lipophagy, where loss of Atg15 is expected to inhibit GFP release from the lipid body marker Erg6-GFP. Methods for lipid quantification and protein analysis are also outlined.

1. 12/12/5 Research Seminar
Search and analysis of factors work on
synthesis and degradation of lipid body
in Saccaromyces cerevisiae
M1 Takeshi Kondo

2. Outline
1, Analysis
ATG15 as a new factor
2, Search
assay system of lipophagy
〜Erg6-GFP processing assay〜

3. Lipid body(LB)
Protein
TAG
(Triacylglycerol)
Sterol
SE
(Sterol ester)
Phospholipid
monolayer
organella storage neutral lipids

4. Autophagy 15
Related Gene

5. Autophagy and Atg15
・essential for degrade subvacuolar vesicles
Autophagosome
・lipase-motief
vacuole
PAS
Atg15
atg15Δ
Autophagic body
U.D. Epple et al. (2001)

6. Strain used in this study
WT
URA3
pRS316
atg15Δ
S332A: Lack the activity
degrade subvacuolar
vesicles
URA3
ATG15
URA3
ATG15S332A
L. N. Nguyen et al. (2011)

7. atg15Δ decrease the number of LD
SD(-Ura)−2days
BODIPY DIC
pRS316/WT
pRS316/atg15Δ

8. BODIPY diffused in vacuole in atg15Δ
SD（-Ura）- BODIPY DIC
2days
pRS316/WT
pRS316/15Δ

9. activity Atg15 is important for normal LD formation
SD(-Ura)-2d BODIPY DIC
WT
atg15Δ
ATG15
ATG15S332A

10. There is no significant difference in the amount of
TAG between WT and atg15Δ
Previous data
0.03
TAG(mg/OD610=1)
0.02
WT
0.01 atg15Δ
N=3
0
0 1 2 3
Time(day)

11. Summary
●Phenotype of atg15Δ
・Dicrease the number of LB
(the size of LB seems to be similar to WT)
・Diffuse the BODIPY in the vacuole
・The amount of TAG is almost same to WT
The activity of Atg15 is imprtant for normal LB formation
A part of TAG isn’t packaged as LD?

12. How Atg15 related to synthesis/degradation of LD
Recycle?
Neutral lipid
synthase vacuole
Neutral
lipid
Atg15
ER lumen
LD
Degradation
products
BODIPY positive neutral lipids?

13. Future plans
1, additional mutation to Atg15S332A
To determine the activity of Atg15 in essential for normal
LB formation/degradation, decrease the activity of Atg15
by additional mutation.
2, Electron microscopic analysis
To check neutral lipids diffuse in vacuole in atg15Δ
3, Visualize vacuole using fluorescence protein
To make it clear that BODIPY diffuse in vacuole in atg15Δ

14. Outline
1, Analysis
ATG15 as a new factor
2, Search
assay system of lipophagy
〜Erg6-GFP processing assay〜

15. lipophagy
LD
vacuole

16. Micro-lipophagy
LD
L: Lipid body
N: nucleus
vacuole V: vacuole
電顕写真
L
N
V

17. Erg6-GFP processing assay
WT Erg6
GFP
vacuole
LD
Western blotting
Erg6-GFP
GFP

18. Erg6-GFP processing assay
pep4Δ Erg6 GFP
vacuole
LD
Erg6-GFP
GFP

19. Erg6-GFP processing assay
Lipophagy deficient mutant
Erg6 GFP
LD
vacuole
Erg6-GFP
GFP

20. Processing assay = “Gyu-don”

21. Speedy Speedy
Cheap Easy to understand
Delicious New

22. Sample preparation for western blotting
Crush using
Suspend by
・Culture beads shocker
100μL lysis buffer
・
sampling
Pellet
(OD=10)
supernatant
Mix with
SDS-sample
buffer
Boil, 3min
Western
blotting
1500g, 20mM Tris-HCl(pH7.9)
4℃, 5min 10mM MgCl2
Prptein 1mM EDTA
concentration 5% Glycerol
measurement Lysis buffer 1mM DTT
0.3M Ammonium Sulfate
Protease inhibitor capsule

23. Erg6-GFP processing assay
YPGly-2d
88
Erg6-GFP
62
47
Pep4
Dependent 35
Processed
bands
GFP 28
24μg each
⇒This system can be used for assay lipophagy

24. Erg6-GFP processing assay
1st 2nd
YPGly-2d
Erg6-GFP
GFP
→Result didin’t reproduce 24μg each

25. Localization of Erg6-GFP
YPGly-2d DIC Erg6-GFP
WT
pep4Δ
2000ms, scale normalized

26. Future plan
●Erg6-GFP processing assay using the strain BY4741
BY4741
Erg6 GFP
pRS315
BY4741Δ
Deletion library

27. Fin.

28. Sample preparation for TAG measurement
●sampling
SD-Trp SD-Trp
1d 2d 3d
Yast pellet
●Preparation for TAG measurement (OD610=15)
measurement
Add buffer Sonication
・Extraction of lipids
・ evaporate (mix)

29. Principle of TAG measurement(kit)
triglyceride
H2O
lipase
COOH Fatty acid
OH
OH glycerol
OH
ATP
Glycerol kinase, Mg2+
ADP
OH
OH
P O2
GPO Dihydroxy acetonephospahate
H2O2
BSDA, aminoantipyrine
peroxydase
H2O
Measurement
Quinoids(Blue pigment) of Absorbance

30. Sample preparation for western blotting
Suspend by Crush by
・Culture 100μL alkaline sonication
・ lysis buffer
sampling
Pellet
(OD=10)
supernatant
Mix with
Neutralization SDS-sample
By 1M HCl buffer
Boil, 3min
Boil, Western
3min blotting
13000rpm,
Prptein
RT, 5min ・0.1M NaOH
concentration
Alkaline ・1% SDS
measurement
Lysis buffer ・Protease inhibitor
capsule