Science - USA (2022-02-18)

larvae showed similar abundance of cysteine,
likely reflecting dietary intake as the main
source of cysteine. By contrast, in fasting
animals, cysteine concentration dropped in
dCTNS−/−larvae (fig. S7E). Conversely,dCTNS
overexpression increased cysteine levels, which
caused a developmental delay similar to cys-
teine treatment (fig. S7, F and G). In sum, our
results indicate that dCTNS recycles cysteine
from the lysosome during fasting.

Cytosolic cysteine synthesis compensates for

defective lysosomal cystine export

To evaluate the contribution of cytosolic cys-

teine synthesis during fasting, we also ana-

lyzed the trans-sulfuration pathway, a process

that requires cystathionineb-synthase (Cbs)

(fig. S8A) ( 17 , 18 ). Depletion ofcbsin the fat

body reduced cysteine levels upon fasting,

but this was not the case in fed animals. Cbs

and dCTNS showed additive roles in main-

taining cysteine concentration in fasted ani-

mals (fig. S8B), suggesting that the phenotypic

effects ofdCTNSdeficiency are partially masked

by the trans-sulfuration pathway. Because the

trans-sulfuration pathway involves the break-

down of methionine, we found thatdCTNS

overexpression increased the concentration

of the intermediates of the trans-sulfuration

pathway, particularly methionine (fig. S8C).

By contrast, methionine was depleted in

Jouandinet al.,Science 375 , eabc4203 (2022) 18 February 2022 3 of 11

Fig. 2. Lysosome-derived cysteine
promotes elevation of TCA cycle
intermediates and antagonizes TORC1
reactivation.(A) Amino acid screen reveals
cysteine as a growth suppressor. Time to
pupariation for larvae fed a low-protein diet
supplemented with the indicated amino acids
all along development (see supplementary
materials and methods for concentrations;
the cysteine concentration is 5 mM). Scale
bar, 1 mm. (B) Cysteine levels in whole
control larvae (lpp>attp40)(N= 5).
(C) Schematic of lysosomal cystine (CySS)
efflux through cystinosin/CTNS.(D) Loss of
Drosophilacystinosin in the fat body
(lpp>dCTNS RNAi, dCTNS-i)leads to higher
TORC1 reactivation upon prolonged fasting.
P-S6K levels in dissected fat bodies from
larvae fasted for the indicated time.lpp>white
RNAi, control background. (E) dCTNS
maintains autophagy during fasting.dCTNS−/−
clones (non-GFP, outlined) in 80-hour AEL
larvae expressing mCherry-Atg8a fasted for
8 hours. Scale bar, 10mm. (F)dCTNS
overexpression suppresses TORC1 activity.
P-S6K levels in dissected fat bodies
from larvae fasted for the indicated time.
Lpp>GFP RNAi (GFP-i), control background.
(G)dCTNSoverexpression induces
autophagy.dCTNSoverexpression clones
(GFP marked, outlined) in 80-hour AEL larvae.
Scale bar, 10mm. (H) dCTNS regulates
TCA cycle intermediate levels upon fasting.
Relative metabolites levels measured
by LC-MS/MS in 80-hour AEL larvae fed or
fasted for 8 hours. Controls are dCTNS+/−
(upper graphs) or GFP-i (lower graphs).
For (B), (D), (F), and (H), data are shown
as mean ± SD. ns,P≥0.05; P≤0.05;
P≤0.01; P≤0.005; ****P≤0.0001
(see the materials and methods for details).

A

Time to pupariation normalized to vehicle

MK TV F L IHWY RGCPQES DNA

1.0

1.5

2.0

Amino acids

Vehicle

Cysteine

FedFasted (6h)

0

1

2

Cysteine (whole body)

Fold change to fed

Cytosol

Cystinosin

(CTNS)

CySS +

CySS

Lysosome

Cys + Cys

Reduction

H

H

+

control

dCTNS-/-

012 4 6 8

P-S6K

S6K

GAPDH

Fasting time (h)

lpp>

controldCTN

S

conroldCTNScontroldCTNScontroldCTNScont

rol

dCTNScontroldCTN

S

Citrate

Cis-aconit

ate aKG

SuccinateFumarate

Malate OAA

control

dCTNS

lpp>

ns ns ns ns ns ns ns **** **** **** ** **** **** ****

*** *** ** * *** *** ** *** *** *** ** *** ***

***

****

Fed

Fasted

dCTNS

overexpression clones

DAPI GFP mCherry-Atg8a/mCherry-Atg8a

dCTNS

• /


• clones

DAPI GFP mCherry-Atg8a/mCherry-Atg8a

Fed

Fasted

BC

D

02468

0

0.5

1

****

***

ns

***

dCTNS

P-S6K/S6K

relative to control

Fasting time (h)

EG

H

0

0.5

1.0

1.5

2.0

Fold change to control

Fed Fasted (8h)

0

1

2

3

4

5

Citrate

Cis-a

coni

tate aKG

SuccinateFumarate

Malate OAA

lpp>

controldCTNS-icontroldCTNS-icontroldCTNS-icontroldCTNS-icontroldCTNS-icontroldCTNS-i

246 8

Fasting time (h)

0 10

P-S6K

S6K

GAPDH

Fasting time (h)

P-S6K/S6K

relative to control

ns

ns

ns *

**

*

0246810

0

1

2

3

dCTNS-i

F

RESEARCH | RESEARCH ARTICLE