121205 リサーチ kondo
TRANSCRIPT
Search and analysis of factors work on synthesis and degradation of lipid body
in Saccaromyces cerevisiae
M1 Takeshi Kondo
12/12/5 Research Seminar
1, AnalysisATG15 as a new factor
2, Searchassay system of lipophagy〜 Erg6-GFP processing assay〜
Outline
TAG (Triacylglycerol)
Protein
Sterol
SE(Sterol ester)
Lipid body(LB)
organella storage neutral lipids
Phospholipid monolayer
Autophagy 15 Related Gene
PASvacuole
Autophagy and Atg15
Atg15
Autophagosome
Autophagic body
・ essential for degrade subvacuolar vesicles
・ lipase-motief
atg15Δ
U.D. Epple et al. (2001)
Strain used in this study
pRS316
URA3
ATG15S332A
URA3
ATG15
URA3
WT
atg15ΔS332A: Lack the activity degrade subvacuolar vesicles
L. N. Nguyen et al. (2011)
pRS316/WT
pRS316/atg15Δ
SD(-Ura)−2days
atg15Δ decrease the number of LD
BODIPY DIC
SD( -Ura) -2days
pRS316/15Δ
pRS316/WT
BODIPY DIC
BODIPY diffused in vacuole in atg15Δ
WT
atg15Δ
ATG15
ATG15S332A
SD(-Ura)-2d
activity Atg15 is important for normal LD formationBODIPY DIC
0 1 2 30
0.01
0.02
0.03
WTatg15Δ
Time(day)
TAG(
mg/
OD6
10=1
)There is no significant difference in the amount of
TAG between WT and atg15Δ
N=3
Previous data
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?
vacuole
How Atg15 related to synthesis/degradation of LD
Atg15
Degradationproducts
ER lumen
LD
Recycle?Neutral lipid synthase
Neutral lipid
BODIPY positive neutral lipids?
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 analysisTo check neutral lipids diffuse in vacuole in atg15Δ
3, Visualize vacuole using fluorescence proteinTo make it clear that BODIPY diffuse in vacuole in atg15Δ
1, AnalysisATG15 as a new factor
2, Searchassay system of lipophagy〜 Erg6-GFP processing assay〜
Outline
lipophagy
LDvacuole
Micro-lipophagy
LD
電顕写真L
N
V
L: Lipid bodyN: nucleusV: vacuolevacuole
LDvacuole
Erg6-GFP processing assay
Erg6GFP
WT
Erg6-GFP
GFP
Western blotting
LDvacuole
Erg6-GFP processing assay
Erg6 GFPpep4Δ
WT pep4Δ
Erg6-GFP
GFP
LDvacuole
Erg6-GFP processing assay
Erg6 GFP
Lipophagy deficient mutant
Lipophagy
Deficient
WT pep4Δ
Erg6-GFP
GFP
Processing assay = “Gyu-don”
Cheap
Speedy
Delicious
Speedy
Easy to understand
New
Sample preparation for western blotting
Pellet(OD=10)
20mM Tris-HCl(pH7.9)10mM MgCl21mM EDTA5% Glycerol1mM DTT0.3M Ammonium SulfateProtease inhibitor capsule
Suspend by 100μL lysis buffer・ Culture・ sampling
Crush using beads shocker
1500g, 4 , 5min℃
supernatant
Prptein concentration measurement
Mix with SDS-sample buffer Boil, 3min
Lysis buffer
Western blotting
YPGly-2d
24μg each
Erg6-GFP processing assay
WTpep4Δ
Erg6-GFP
GFP
Pep4 DependentProcessedbands
88
62
47
35
28
⇒This system can be used for assay lipophagy
Erg6-GFP processing assay
24μg each
Erg6-GFP
GFP
YPGly-2d
1st 2nd
→Result didin’t reproduce
WT
pep4Δ
DIC Erg6-GFPYPGly-2d
2000ms, scale normalized
Localization of Erg6-GFP
Future plan
●Erg6-GFP processing assay using the strain BY4741
pRS315
GFPErg6
BY4741
BY4741Δ
Deletion library
Fin.
Sample preparation for TAG measurement
SD-Trp SD-Trp
Yast pellet(OD610=15)
measurement
●sampling
●Preparation for TAG measurement
Sonication(mix)
1d 2d 3d
・ Extraction of lipids・ evaporate
Add buffer
Principle of TAG measurement(kit)
triglyceride
lipaseFatty acid
OHOHOH
COOH
H2O
ATP
ADPGlycerol kinase, Mg2+
OHOH
P O2
Dihydroxy acetonephospahate
H2O2
peroxydase
Quinoids(Blue pigment)Measurement of Absorbance
glycerol
H2O
BSDA, aminoantipyrine
GPO
Sample preparation for western blotting
Pellet(OD=10)
・ 0.1M NaOH・ 1% SDS・ Protease inhibitor capsule
Suspend by 100μL alkalinelysis buffer
・ Culture・ sampling
Crush by sonication
supernatant
Prptein concentration measurement
Mix with SDS-sample buffer Boil, 3min
Alkaline Lysis buffer
Western blotting
NeutralizationBy 1M HCl
Boil,3min
13000rpm,RT, 5min