fig. S5, G, and H), which indicated that it is indeed the loss of GATOR1 proteins that is driving
aberrant m TORC1 signaling in these cells.
The proliferation of the GATOR1-null cancer
cells was very sensitive to the m TORC1 inhibitor
rapamycin, with median inhibitory concentration
(IC50) values in the 0.1 to 0.4 nM range (Fig.
5G). These values are many orders of magnitude
less than for cell lines that are not considered
rapamycin-sensitive, like HeLa and HT29 cells,
and at the low end of cancer cell lines, like PC3
and Jurkat cells, which have lost PTEN function
(24–26), an established negative regulator of the
m TORC1 pathway. In addition, the forced expression of DEPDC5 in the MRKNU1 (DEPDC5−/−)
cell line led to a marked reduction in its proliferation (fig. S5J).
In conclusion, we identify the octomeric
GATOR complex as a critical regulator of the
pathway that signals amino acid sufficiency to
m TORC1 (Fig. 5G). The GATOR1 subcomplex
has GAP activity for RagA and RagB and its loss
makes m TORC1 signaling insensitive to amino
acid deprivation. Inactivating mutations in GATOR1
are present in cancer and may help identify tu-
mors likely to respond to clinically approved
pharmacological inhibitors of m TORC1.
References and Notes
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Acknowledgments: We thank all members of the Sabatini
lab for helpful suggestions, E. Spooner for the mass
spectrometric analysis of samples, and N. Kory for technical
assistance. This work was supported by grants from the
NIH (CA103866 and AI47389) and Department of Defense
(W81XWH-07-0448) to D.M.S. and the National Cancer
Institute (NIH) (U24CA143867) to M.M. and awards from
the David H. Koch Graduate Fellowship Fund to L.B.-P.; the
NSF Graduate Research Fellowship Program to L.C.; the
Harvard-MIT Health, Sciences, and Technology IDEA2 program
to W. W.C.; and the American Cancer Society to B.C.G. D.M.S. is
an investigator of the Howard Hughes Medical Institute.
Materials and Methods
Figs. S1 to S5
26 October 2012; accepted 5 April 2013
Impaired a-TTP-PIPs Interaction
Underlies Familial Vitamin E Deficiency
Nozomu Kono,1,2 Umeharu Ohto,1 Tatsufumi Hiramatsu,1 Michiko Urabe,1 Yasunori Uchida,1
Yoshinori Satow,1 Hiroyuki Arai1,2†
a-Tocopherol (vitamin E) transfer protein (a-TTP) regulates the secretion of a-tocopherol from
liver cells. Missense mutations of some arginine residues at the surface of a-TTP cause severe
vitamin E deficiency in humans, but the role of these residues is unclear. Here, we found that
wild-type a-TTP bound phosphatidylinositol phosphates (PIPs), whereas the arginine mutants
did not. In addition, PIPs in the target membrane promoted the intermembrane transfer of
a-tocopherol by a-TTP. The crystal structure of the a-TTP–PIPs complex revealed that the
disease-related arginine residues interacted with phosphate groups of the PIPs and that the PIPs
binding caused the lid of the a-tocopherol–binding pocket to open. Thus, PIPs have a role in
promoting the release of a ligand from a lipid-transfer protein.
Intracellular lipid transport is required for nu- merous cellular events (1, 2). Lipids are transported between organelles by vesicles
or are delivered by lipid-transfer proteins (3).
Some lipid-transfer proteins possess specific
organellar-targeting motifs to assure precise lipid
transport from donor to acceptor organelles (1, 2).
However, many other lipid-transfer proteins have
no known organellar-targeting domains, and the
molecular bases underlying intracellular lipid transport by these proteins are largely unknown.
1Graduate School of Pharmaceutical Sciences, University of
Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan. 2Core
Research for Evolutional Science and Technology, Japan Science
and Technology Agency, 4-1-8 Honmachi, Kawaguchi, Saitama,
*These authors contributed equally to this work.
†Corresponding author. E-mail: firstname.lastname@example.org
a-Tocopherol transfer protein (a-TTP), which
specifically binds a-tocopherol (a-Toc), the most
abundant form of vitamin E in mammals, is ex-
pressed in the liver where it regulates the amount
of a-Toc secreted into the plasma (4). Heritable
mutations in the a-TTP–encoding gene result in
ataxia with vitamin E deficiency (AVED), an au-
tosomal recessive disorder associated with low
circulating vitamin E concentrations and neuro-
degenerative pathology (5). More than 20 muta-
tions in the a-TTP gene have been identified in
AVED patients (5). a-TTP is a member of the
Sec14-like protein family (6) and has a lipid-
binding domain, the Sec14 domain. Because
a- TTP has no known organellar-targeting domain,
we focused on the mutations in AVED patients to
investigate the molecular mechanism underlying
a-TTP–mediated a-Toc transport in liver cells.
Of the nine disease-associated missense muta-
tions (Fig. 1A), three (R59W, R192H, and R221W)
are located in one region on the a-TTP protein
surface, which is distinct from the a-Toc binding
site (7) (Fig. 1B). The R59W and R221W mu-
tations give rise to the severe, early-onset form of
the disease (8), indicating that these arginine res-
idues are critical for a-TTP function.