Rag GTPases - Ras Superfamily Small G Proteins: Biology and Mechanisms 2 - page 273

mTORC1) interacting proteins through an immunoprecipitation-mass spectromet-
ric analysis approach (Kim et al. 2008 ; Sancak et al. 2008 ).
Similar to other small GTPases, the activation state of the Rag GTPases is
controlled by their guanine nucleotide state, and this is proposed to be regulated
by amino acids (Sancak et al. 2008 ). Specifically, in the presence of amino acids,
the heterodimeric Rag complex is active, where RagA and RagB are GTP bound
and RagC and RagD are GDP bound. Alternatively, in the absence of amino acids,
RagA and RagB are GDP bound and RagC and RagD are GTP bound. Consistently
in yeast, amino acid-rich conditions promote the active form where Gtr1 GTP
bound is in complex with Gtr2 GDP bound (Fig. 12.1 ) (Binda et al. 2009 ). It
appears that RagA/B may play a more dominant role over RagC/D in the activation
of mTORC1 by amino acids in mammals. RagA/B binds directly to mTORC1
(Gong et al. 2011 ), and overexpression of constitutively active RagA/B (GTP
bound) alone renders mTORC1 insensitive to amino acid starvation (Kim
et al. 2008 ; Sancak et al. 2008 ). Unlike mammals, active Gtr1 (GTP bound) can
only partially activate TORC1 (Binda et al. 2009 ). Co-expression of RagC/D
enhances mTORC1 activation, in part because RagC/D stabilizes RagA/B. In
fact, the depletion of RagA/B significantly diminished RagC/D levels, consistent
with the notion that RagA/B stabilizes RagC/D by forming heterodimers (Sancak
et al. 2008 ; Sekiguchi et al. 2001 ). Furthermore, the inactive mutant Rag complex
(RagA/B GDP bound and RagC/D GTP bound) fails to respond to amino acids and
restrains mTORC1 activity even under amino acid sufficiency (Kim et al. 2008 ;
Sancak et al. 2008 ). These findings suggest that the presence of amino acids
determines the guanine nucleotide state of the Rag GTPases and ultimately
mTORC1 activation. However, it is worth noting that a new study using different
biochemical methods revealed that amino acids activate mTORC1 without altering
the guanine nucleotide loading of the Rag GTPases (Oshiro et al. 2014 ). Thus,
whether amino acids regulate the guanine nucleotide loading of the Rag GTPases is
currently under debate.
12.2.1 Rag GTPases at the Lysosome
Rag GTPases appear to reside at the lysosome in order to activate mTORC1.
Whether or not the Rags can localize to other cellular compartments in response
to stimuli is unknown. The active Rag complex (RagA/B GTP bound and RagC/D
GDP bound) in amino acid sufficient conditions binds directly to mTORC1 via
interaction with raptor (an essential subunit of mTORC1) and recruits it to the
lysosome. How mTORC1 translocates to the lysosome is unknown. Some speculate
that the Rag GTPases may come off at some point to retrieve mTORC1, but there is
no data to support this. When amino acids are limiting, mTORC1 is scattered
throughout the cell at undefined locations, but it is quickly redistributed to vesicles
containing lysosome-associated membrane protein 2 (LAMP2; a lysosome marker)
and RAB7 (late endosome marker) in response to amino acid stimulation (Sancak