Science - USA (2022-02-18)

(fig. S3B). Depletion of PC also suppressed
TORC1 reactivation during fasting (Fig. 1D),
supporting the functional link between the
TCA cycle and TORC1 signaling. These data
suggest that at the onset of fasting, TORC1

inhibition appears necessary to raise the con-

centration of TCA cycle intermediates, which

in turn affect TORC1 reactivation, possibly

through the synthesis of building blocks down-

stream of PC, i.e., cataplerosis.

Cysteine suppresses growth in a

TORC1-dependent manner

In the second screen, we tested the effects of

supplementation with single amino acids on

growth of larvae fed a low-protein diet (see the

supplementary materials) (Fig. 2A). Fasting

and a low-protein diet may trigger distinct

metabolic states with intrinsic differences, but

both protocols trigger a starvation response.

Supplementation of food with cysteine sup-

pressed growth in larvae to a distinctly greater

extent than any other amino acid, and this

phenotype was accentuated in a low-protein

diet (see the supplementary text S1 and fig. S4,

A to D). The inhibition of growth by cysteine

was interdependent with TORC1 signaling,

because cysteine treatment partially inhibited

TORC1 activity in fat bodies and constitutive

activation of TORC1 in GATOR1-null (nprl2−/−)

mutants ( 12 ) and partially restored growth

under cysteine supplementation (fig. S5, A

to E). Conversely, cysteine supplementation

did not further suppress growth in mutants

with constitutive suppression of TORC1 activ-

ity caused by either loss of GATOR2 (mio−/−)

or overexpression of TSC complex subunits

(lpp>UAS-TSC1, UAS-TSC2)inthefatbody

(fig. S5, E and F). Altogether, these data sug-

gest that cysteine has a specific inhibitory

effect on growth and TORC1 signaling in

larvae fed a low-protein diet, and that this

growth suppression requires suppression of

TORC1 signaling.

Lysosomal cystine transporter dCTNS regulates

cysteine level during fasting

The results of our two screens led us to ex-

plore the relationship among cysteine metab-

olism, the TCA cycle, and TORC1 during fasting.

Cysteine concentrations increased during fast-

ing (Fig. 2B and fig. S6A) ( 13 ), and cysteine

treatment increased the concentration of TCA

cycle intermediates, particularly for animals

fed a low-protein diet (fig. S6B). To further

understand this effect of cysteine, we searched

for its intracellular source during fasting.

Abolishingautophagyinthefatbodydecreased

cysteine levels upon fasting (fig. S7A), suggesting

that autolysosomal function regulates cysteine

balance. Thus, we focused on the role of the

lysosomal cystine transporter cystinosin in re-

cycling cysteine during fasting. Cystinosin,

which is encoded byCTNSin mammals, is

mutated in the lysosomal storage disorder

cystinosis and has been implicated in the reg-

ulation of TORC1 signaling and autophagy

( 14 Ð 16 ). Endogenous tagging of theDrosophila

orthologCG17119(hereafter referred to as

dCTNS) confirmed its specific lysosomal locali-

zation in cells of the fat body (fig. S7, B and

C).dCTNS−/−larvae showed accumulation

of cystine (fig. S7D), consistent with a role for

cystinosin in lysosomal cystine transport (Fig.

2C). In fed conditions, control anddCTNS−/−

Jouandinet al.,Science 375 , eabc4203 (2022) 18 February 2022 2 of 11

1.5

1

0

0.5

-1.5

-1

-0.5

A Starvation time (h)

P-S6K

S6K

GAPDH

01234 56 7 8 kDa

70

70

35

P-S6K/S6K

normalized to fed

(^02468)

0. 
   


1. 5


2. 0

Starvation time (h)

Whole larvae

3

2

0

1

-3

-2

-1

Starvation time (h)

062 4 8 06

Starvation time (h)

Fat body

Fold change to fed

********

****

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

****

********

ns****

********

**

0 2 4 6 8

0

1

2

3

4

Citrate

0 2 4 6 8

0

1

2

3

4

Isocitrate

Starvation time (h)

Itaconic acid

Citraconic acid

Glutamine

aKG

Orotate

Aconitate

Citrate

Succinate

Malate

Maleic acid

Isocitrate

Carnitine

3-phospho-serine
AICAR
Geranyl-PP
Glycerophosphocholine

Methylmalonate

Cholesteryl sulfate

3-phosphoglycerate

Phosphoenolpyruvate

Fumarate

2-keto-isovalerate

AICAR

SAM

Itaconic acid

Citraconic acid

aKG

Glutamine

Phenylpropiolic acid

2,3-diphosphoglycerate

Quinolinate

N-acetylspermine

Carbamoyl phosphate

3-phosphoglycerate

Pipecolic acid

UTP

AT P

DGTP

CTP

Acetylcarnitine

Glutamate

UDP-D-glucoronate

Icocitrate

Citrate

3-phospho-serine

GTP

Aconitate

UDP-D-glucose

Guanosine 5-DP,3-DP

Citrate

Isocitrate

Succinate aKG

Malate

OAA

Pyruvate

Fumarate

PC

Glucose/lactate/alanine

P-S6K

S6K

0

Starvation time (h)

2 4 6 8 10.5

lpp>

attp4

0

pcb-iattp40pcb-iattp4

0

pcb-iattp4

0

pcb-iattp40pcb-iattp40pcb

-i

Starvation time (h)

*** *** ***

****

ns

02468

0.0

0.5

1.0

pcb RNAi

P-S6K/S6K

normalized to control

B

0 6 0 6

**

**

0

2

4

6

8

0

2

4

6

8

10

Citrate Isocitrate

CD

Fig. 1. TORC1 reactivation upon prolonged fasting correlates with increase of TCA cycle intermediates.
(A) Prolonged fasting leads to TORC1 reactivation. Phosphorylation levels of the direct TORC1 target
S6K in dissected fat bodies from fasting larvae (i.e., placed on a tissue soaked in PBS). (B) Heatmap
metabolite levels (LC-MS/MS) from whole mid-third-instar larvae (left) or dissected fat bodies (right), fed
(0h) and fasted. Lower panels are individual plots from the same dataset. (C) Schematic of anaplerosis
through PC. (D) Knockdown ofpcb/PC suppresses mTORC1 reactivation upon prolonged fasting. For (B) and
(D), data are shown as mean ± SD. ns,P≥0.05; **P≤0.01; *P≤0.005; **P≤0.0001 (see the
materials and methods for details).

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