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