We used electrophoresis and mass spectrometry
analysis to exclude major proteolytic degradation during the preparation and crystallization
process (fig. S5). Thus, we could not see residues
21 to 80, probably because of flexibility in the
crystal. LAMTOR1 discontinued at the N and C
termini, leaving an apparently accessible space
on the solvent-exposed side of the LAMTOR2
and LAMTOR3 heterodimer. The a2 helices of
both LAMTOR2 and LAMTOR3 are thought to
mediate interaction with other proteins (12, 13).
The pentameric structure showed that the absent
C-terminal helices of LAMTOR4 and LAMTOR5
were spatially complemented by helices a4 and
a5 of LAMTOR1 (Fig. 1B), closely resembling the
interaction mode observed for Ego2 and Ego1
(15) (fig. S4). The structurally resolved region of
LAMTOR1 is largely helical and stabilizes the
complex by forming a U-shaped belt around the
two heterodimers, providing additional contacts
that may contribute to increased affinity between the different subunits (Fig. 2). We identified three areas of contact between LAMTOR1
and LAMTOR3, LAMTOR4, or LAMTOR5 (Fig. 2,
figs. S6 and S7, and supplementary text).
To investigate the physiological relevance of
the extended LAMTOR1 conformation in the pentameric complex, we generated truncations and
alanine mutants to specifically abolish the interaction to each of the remaining LAMTOR subunits (Fig. 3A). The choice of mutated residues was
based on structurally identified contacts (Fig. 2
and fig. S1). For instance, in LAMTOR1_LT2*.SH,
we mutated V148 and D149 of LAMTOR1 to alanine. LAMTOR1 variants were tagged with a
streptavidin-binding peptide and a hemagglutinin
(HA) epitope, hereafter designated as an SH tag.
LAMTOR1WT.SH and mutants, as well as SH.GFP
(GFP, green fluorescent protein) as a control, were
expressed in modified human embryonic kidney
(HEK) 293 Flp-In T-REx cells (hereafter HEK293)
(Fig. 3B). LAMTOR1WT.SH coimmunoprecipitated
all other LAMTOR proteins, Rags, SLC38A9 (the
lysosomal amino acid transporter), and components of the BORC (for BLOC1–related complex):
namely, Snapin, C10orf32, and C17orf59 (17–21).
The majority of mutants neither formed pentameric complexes nor interacted with their known
partners. LAM TOR1_1-147.SH associated weakly
with the remaining LAMTOR subunits. LAMTOR1_
L T2*. SH mutations interfered with complex stability but permitted association with the Rags. In
contrast, LAMTOR1_1-150.SH associated with the
remaining LAMTOR and BORC components but
did not recruit either Rags or SLC38A9. Thus,
the belt-like function of LAM TOR1 appears to be
fundamental for LAMTOR stability, and the C
terminus (residues 150 to 161) of LAMTOR1 is
essential for association with the Rags.
We then generated a LAMTOR1 hypomorph cell
line (hereafter LAMTOR1HM) (fig. S8 and supplementary text) and transiently transfected it with
LAMTOR1WT.SH and the same LAMTOR1 mutants previously tested in HEK293 cells. They all
colocalized with endogenous LAMP1 (
lysosomal-associated membrane protein 1) (fig. S8C), indicating
that differences observed in the interactome analysis were not due to mislocalization. To functionally
address the role of the C terminus of LAMTOR1 in
anchoring the Rags to the lysosomal surface (3),
we established LAMTOR1HM cell lines stably expressing LAMTOR1WT.HA, LAMTOR1_1-106.HA,
or LAMTOR1_1-150.HA (fig. S9A). LAMTOR1WT.HA
restored the interaction with the Rags and SLC38A9
(fig. S9B), whereas LAMTOR1_1-150.HA did not. In
wild-type cells, RagC was recruited to LAMP1 structures (Fig. 3C). LAMTOR1 deletion resulted in impaired recruitment of RagC that could be restored
by expression of LAMTOR1WT.HA but not by
Complementarily, we observed colocalization of
endogenous RagC with LAMTOR1WT.HA but not
with LAMTOR1_1-106.HA or LAMTOR1_1-150.HA
(fig. S9, C and D). Next, we tested the cell lines for
their signaling properties (Fig. 3D). Control cells
responded to withdrawal of amino acids with low
phosphorylation of both p70S6K (ribosomal protein S6 kinase beta-1) and downstream S6. Reexposure of control cells to all essential amino acids
and glutamine readily increased phosphorylation
of p70S6K and S6, indicating m TORC1 activation.
LAMTOR1HM cells showed impaired p70S6K and
S6 phosphorylation when reexposed to amino
acids, and this defect was rescued by expression of
LAMTOR1WT.HA but not by LAMTOR1_1-150.HA.
Although defective in mTORC1 signaling,
LAMTOR1_1-150.HA cells contained stable pentameric LAMTOR complexes (Fig. 3D). Thus, the C
terminus of LAMTOR1 appears to be required
to recruit the Rags, thereby controlling amino
acid–dependent activation of m TORC1. The 12 C-terminal residues of LAMTOR1 are highly conserved and contain two prominent charged (KEE)
and hydrophobic (LVV) clusters (Fig. 3 and fig.
S1), which we next mutated to alanine triplets.
LAMTOR1_KEE.SH and LAMTOR1_LVV.SH interacted with LAMTOR3 and LAMTOR2, but
mutation of KEE impaired the association with
the Rags, and LVV mutation completely abolished
it (Fig. 3E).
The CTD of Gtr1 associates with Ego1 and
Ego2 (22), and dimerized CTDs of the Rags are
necessary for LAMTOR1 binding (23). The termini
380 20 OCTOBER 2017 • VOL 358 ISSUE 6361
Fig. 4. Interaction of Rag GTPases with the LAMTOR complex. (A) Crystal structure of the heptamer.
Dark green, RagA CTD; light green, RagC CTD; red, LAMTOR1; purple, LAMTOR2; blue, LAMTOR3; orange,
LAMTOR4; yellow, LAMTOR5. N and C termini are labeled. (B) Rotated view of the structure shown in
(A) with the G domains, modeled on the basis of the Gtr1 and Gtr2 structures [PDB: 4ARZ (24)] to define
their positional orientation in the heptameric complex. Experimental cross-links are indicated by black
dotted lines. A cartoon representation is included (bottom right). The antiparallel arrows between two
roadblock domains indicate the central b-sheet augmentation of roadblock heterodimers.