分裂酵母における細胞壁ホメオスタシスの化学遺伝学的解析
P-20

西村 慎一
東京大学大学院農学生命科学研究科

Summary

ORFeome and localizome

Fungal cell wall is one of the drug targets, yet the detailed molecular mechanism
remains to be clarified. Theonellamides (TNMs) are marine sponge-derived peptides,
targeting membrane sterols. They bind to cholesterol in mammalian cells, andergosterol in yeast cells [1,2]. In fission yeast, TNMs induce accumulation of cell wallmaterials, in a Rho1- and Bgs1-dependent manner [1]. This phenomenon is unique toTNMs, and is not induced by traditional polyene antifungals. For understanding themolecular mechanisms of cell wall biosynthesis, we are investigating the molecularmechanism of the antifungal action of TNMs. To identify proteins whose subcellular
localization is affected by TNM, and hopefully involved in the mode of action of TNM,
we utilized the Localizome database which was established based on the cloned fission
yeast ORFeome [3,4]. We focused on ~400 proteins, which were annotated to belocalized at ‘periphery’ or ‘cell tip and site of septum formation’ in the Localizomedatabase. Cellular localization of more than 100 proteins was changed by TNM-A; ~30
proteins were recruited to the sites with thick cell wall, while ~30 proteins disappeared
from the plasma membrane to form cytoplasmic dots. In this presentation, we will show
the detail of the screening results and would like to discuss the functional relationships
between ergosterol and cell wall biosynthesis.

S. pombe genome
ORFeome collections
ORF-YFP

Localizome
ORF library

Mitochondria

Periphery

Microtubule

Cytosol

Nucleus

ER[1] Nishimura, S. et al. Nat. Chem. Biol. 2010, 6, 519. [2] Arita, Y. et al. Chem. Biol. 2015, 22, 604. [3]Matsuyama, A. et al. Nat. Biotechnol. 2006, 24, 841. [4] Shirai, A. et al. J.
Biol. Chem. 2008, 283, 10745.

I. Sterol binding antifungal, theonellamide A (TNM-A).
ab

DMSO

III. Proteins incorporated or not correctly delivered.

TNM-A (2 μg/ml, 2 h)aGene ID

Gene name

SPAC15A10.09c
SPBC651.03c
SPCC757.11c
SPBC1773.15
SPAC1002.16c
SPCC320.08
SPCC417.10
SPBC15C4.04c
SPBC887.17
SPAC1B3.15c
SPAC1F8.03c

pun1
gyp10

SPBC4B4.13c
SPAC1B3.16c
SPAC922.08c
SPBC1271.10c
SPBC16A3.17c
SPBC649.04
SPAC4G9.07
SPBC359.03c
SPAC1399.01c
SPBC13A2.04c
SPAPB1A11.01
SPAP7G5.06

liz1
vht1
cat1

dal52

dal51

str3

uvi15
mug133
aat1
ptr2
mfc1
per1

Molecular
WeightNo. of TMDomain

Gene Product

31.4
42.5
51.9
56
55.2
56.7
56.6
59.7
68.5
69
70.5

SUR7 family protein Pun1 (predicted)
GTPase activating protein Gyp10transmembrane transporter (predicted)
dipeptide transmembrane transporter Dal5h2 (predicted)
carboxylic acid transmembrane transporter (predicted)
transmembrane transporter (predicted)
dipeptide transmembrane transporter Dal5h1 (predicted)
amino acid transmembrane transporter (predicted)
nucleobase transmembrane transporter (predicted)
transmembrane transporter (predicted)
plasma membrane siderophore-iron transmembrane transporter Str3

4
1
1210
1010
1211
10
11
13

57.9
62.8
63.6
65.2
65.2
9.3
60.3
63.2
64.5
68.4
55.3
64

plasma membrane pantothenate transmembrane transporter Liz1
plasma membrane vitamin H transmembrane transporter Vht1
plasma membrane arginine/lysine amino acid transmembrane transporter Cat1transmembrane transporter (predicted)
transmembrane transporter (predicted)
tail anchored plasma membrane protein Uvi15
UPF0300 family protein 2
plasma membrane amino acid transmembrane transporter Aat1
nucleobase transmembrane transporter (predicted)
plasma membrane PTR family peptide transmembrane transporter Ptr2
prospore membrane copper transmembrane transporter Mfc1
plasma membrane amino acid transmembrane transporter Per1

1012111113111210
1211IV. Proteins accumulated at cell tips and/or septa.

cTNM
heronamide
Bgs1
lipid raft

Gene nameMolecularWeightdma1
fic1
mug86
mug33
git3
mug170rga730.530.833.93753.8
48.3
76.6

SPBP8B7.27
SPCC1020.01c
SPAC607.10
SPBC27.04
SPAC631.01c
SPBC16E9.11c
SPAC11E3.02c
SPAC6F12.12
SPBC32H8.13c

mug30
pma2
spo3
uds1
acp2
pub3
ync13
par2
mok12

93.4
110.1
119.4
118.2
29.8
89.2
142.9
72.4
266.5

increased polarity

Rho1

No. of TMDomain

Gene Productmitotic spindle checkpoint ubiquitin ligase Dma1
C2 domain protein Fic1
plasma membrane acetate transmembrane transporter (predicted)
Tea1-interacting protein involved in exocytosis
G-protein coupled receptor Git3arrestin family Schizosaccharomyces specific protein Mug170
RhoGAP, GTPase activating protein Rga7
HECT-type ubiquitin-protein ligase E3, found in association with TRAMP complex(predicted)
P-type proton ATPase, P3-type Pma2
sporulation protein Spo3
septation protein Uds1
F-actin capping protein beta subunit Acp2
HECT-type ubiquitin-protein ligase E3 Pub3 (predicted)
Munc family exocytic/endocytic regulator Ync13
protein phosphatase PP2A regulatory subunit B-56 Par2
prospore membrane alpha-1,3-glucan synthase Mok12aBgs1

Rho1

Gene IDSPAC17G8.10c
SPBC83.18c
SPAC5D6.09c
SPCC1739.10
SPCC1753.02c
SPCP1E11.03
SPBC23G7.08c

septun to cell tip

low polarity-> accumulation

647914b
ORF-YFP / Cfw

others

TNM-A

10 μm

DMSO

tdTom-Bgs1 / calcofluor white

DMSO

css1-2

10 μm

10 μm

TNM-A

TNM-A (2 μg/ml, 2 h)

II. Summary of the localizome screening.
S. pombe proteome(5,064)

Notes.

periphery (348)cell tip and site of septum formation(129)
Localizome database
263

85

44

group 4(29)110
effect of TNM

group 3(9)group 1(35)group 2(37)This research was conducted with Mr. Akifumi Suganaga, Dr. Akihisa Matsuyama, andProf. Minoru Yoshida. Cell wall analysis is being carried out in collaboration with Dr.
Masako Ohsumi, Dr. Yoshihisa Namase, Ms. Vanessa S. D. Carvalho, Dr. Juan C. G.
Cortés, and Dr. Juan. C. Ribas. The natural product is supplied by Prof. Shigeki
Matsunaga.