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
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