E. coli IscU was overproduced and isolated in its
apo form, and its [4Fe-4S] cluster was then
reconstituted using previously established methods (44). Indeed, when E. coli IscU containing a
[4Fe-4S] cluster was included in excess of LipA,
additional turnover was observed (Fig. 4B). The
cochaperones HscA and HscB, encoded within the
isc gene operon, function in facilitating cluster
transfer from E. coli IscU to its recipient proteins,
including NfuA (37, 45). However, no increase in
rate or product formation was observed when
HscA, HscB, MgCl2, and adenosine triphosphate
(ATP) were included in the reaction with holo IscU
or holo NfuA; in fact, the inclusion of HscA and
HscB was slightly inhibitory (Fig. 4, C and D).
We also carried out a similar scrambling study
of the effect of IscU on LipA catalysis. When the
reaction was conducted with unlabeled IscU and
unlabeled LipA in the presence of 1 mM Na234S,
almost all of the resulting lipoyl product contained
two 32S atoms; very little contained the mixed 32S/
34S or the 34S/34S product (Fig. 4E). The effect of
citrate on the enhancement of the LipA reaction
by IscU could not be studied because IscU’s cluster
was unstable under those conditions (fig. S4).
The resistance to the idea that an Fe-S cluster
can act as a sulfur source during the radical-mediated sulfhydrylation of unactivated carbon
centers is largely due to the consequence of the
enzyme inactivating itself after only one turnover.
Our finding that E. coli NfuA or IscU can reinstall
the Fe-S cluster in E. coli LipA after each turnover,
in a process that is not rate-limiting, suggests that
this concern is no longer warranted. Our studies
also most likely explain why patients with defects
in NFU1, the mammalian ortholog of NfuA, display phenotypes that are consistent with lipoic
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We thank B. Wang for assistance with the synthesis of 34S-labeled
sulfide, and P. Babitzke (Penn State) for E. coli BW25113. Supported
by NIH grant GM122595 and NSF grant MCB-1158486 (S.J.B.). S.J.B.
is an Investigator of the Howard Hughes Medical Institute.
Materials and Methods
Figs. S1 to S4
Tables S1 and S2
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18 April 2017; accepted 6 September 2017
Crystal structure of the human
lysosomal mTORC1 scaffold complex
and its impact on signaling
Mariana E. G. de Araujo,1 Andreas Naschberger,2*† Barbara G. Fürnrohr,2*
Taras Stasyk,1 Theresia Dunzendorfer-Matt,2 Stefan Lechner,2 Stefan Welti,2
Leopold Kremser,3 Giridhar Shivalingaiah,2 Martin Offterdinger,4 Herbert H. Lindner,3
Lukas A. Huber,1,5‡ Klaus Scheffzek2‡
The LAMTOR [late endosomal and lysosomal adaptor and MAPK (mitogen-activated protein
kinase) and m TOR (mechanistic target of rapamycin) activator] complex, also known as
“Ragulator,” controls the activity of m TOR complex 1 (m TORC1) on the lysosome. The crystal
structure of LAMTOR consists of two roadblock/LC7 domain–folded heterodimers wrapped
and apparently held together by LAMTOR1, which assembles the complex on lysosomes. In
addition, the Rag guanosine triphosphatases (GTPases) associated with the pentamer through
their carboxyl-terminal domains, predefining the orientation for interaction with m TORC1.
In vitro reconstitution and experiments with site-directed mutagenesis defined the
physiological importance of LAMTOR1 in assembling the remaining components to ensure
fidelity of m TORC1 signaling. Functional data validated the effect of two short LAMTOR1 amino
acid regions in recruitment and stabilization of the Rag GTPases.
The LAMTOR [late endosomal and lysosomal adaptor and MAPK (mitogen-activated pro- tein kinase) and m TOR (mechanistic target of rapamycin) activator] complex is a pen- tameric complex [LAMTOR1 to LAMTOR5;
also known as p18, p14, MP1 (MEK binding part-
ner 1), C7orf59, and HBXIP (hepatitis B virus
X-interacting protein)] on late endosomes and
lysosomes (1). LAMTOR2 and LAMTOR3 scaf-
fold MEK1 (MAPK kinase 1) and ERK1 or ERK2
(Ras-dependent extracellular signal–regulated ki-
nase) to lysosomes, providing spatial and temporal
specificity in the MAPK pathway (2). Furthermore,
LAMTOR anchors the Rag guanosine triphospha-
tases (GTPases) to the lysosomal surface (3) and
may serve as a guanine nucleotide exchange fac-
tor (GEF) toward the Rag proteins (i.e., the “Rags”),
sciencemag.org 20 OCTOBER 2017 • VOL 358 ISSUE 6361 377
1Division of Cell Biology, Biocenter, Medical University of
Innsbruck, 6020 Innsbruck, Austria. 2Division of Biological
Chemistry, Biocenter, Medical University of Innsbruck, 6020
Innsbruck, Austria. 3Division of Clinical Biochemistry,
Biocenter, Medical University of Innsbruck, 6020 Innsbruck,
Austria. 4Division of Neurobiochemistry-Biooptics, Biocenter,
Medical University of Innsbruck, 6020 Innsbruck, Austria.
5Austrian Drug Screening Institute, 6020 Innsbruck, Austria.
*These authors contributed equally to this work. †Present address:
Division of Genetic Epidemiology, Medical University of Innsbruck,
6020 Innsbruck, Austria. ‡Corresponding author. Email: lukas.a.
firstname.lastname@example.org (L.A.H.); email@example.com (K.S.)
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