Rag GTPases - Ras Superfamily Small G Proteins: Biology and Mechanisms 2 - page 276

12.2.3 Vacuolar H + -ATPase
The vacuolar H + -ATPase (v-ATPase) controls mTORC1 activation in response to
amino acids in both Drosophila melanogaster and mammalian cells through inter-
action with Rag GTPases and Ragulator on the lysosome surface (Zoncu
et al. 2011a ). The v-ATPase pumps protons into lysosomes to maintain the acidic
lysosomal pH and function. Two domains comprise the v-ATPase: the peripheral
cytosolic V 1 domain (containing eight subunits: A-H) and the integral membrane
V 0 domain (has five subunits: a, d, c, c 0 ,c 00 ). The V 1 domain hydrolyzes ATP to
drive proton translocation from the cytosol through the V 0 channel into the lyso-
some, acidifying it (Nishi and Forgac 2002 ).
Both the V 1 and V 0 domains interact with the Ragulator, whereas only the V 1
domain binds to the Rag GTPases. These interactions are thought to be sensitive to
amino acid availability, like Rag GTPase-Ragulator binding. For example, the
interaction between v-ATPase V 1 domain and the Ragulator and Rag GTPases is
regulated by amino acids, strengthened by amino acid starvation, and weakened by
amino acid stimulation. Chemical inhibition of the v-ATPase with Salicylihalamide
A renders the V 1 domain-Rag and V 1 domain-Ragulator associations insensitive to
amino acids (Zoncu et al. 2011a ). In addition, chemical inhibition of the v-ATPase
also desensitizes Rag GTPase-Ragulator binding to amino acid stimulation,
suggesting that the v-ATPase is upstream of the Ragulator (Fig. 12.1 )(Bar-Peled
et al. 2012 ). Unlike the Ragulator, knockdown of v-ATPase subunits or v-ATPase
chemical inhibitors do not affect Rag lysosomal localization. Despite having no role
on lysosomal Rag localization, the knockdown of v-ATPase subunits precludes
mTORC1 lysosomal localization and activation. ATP hydrolysis by the v-ATPase
V 1 domain is important for Rag-mTORC1 interaction; however, there is currently no
mechanistic explanation as to why this is the case (Zoncu et al. 2011a ).
Although many unanswered questions remain, a recent model has emerged
which proposes that amino acids within the lysosomal lumen communicate to the
v-ATPase through an “inside-out” mechanism (an increase of amino acids inside
the lysosomal lumen signals to and activates mTORC1, which resides on the
outside of the lysosome; Fig. 12.1 ). The v-ATPase, through contact interactions,
controls the Ragulator that anchors the Rag GTPases to the lysosome. When the
Rag complex is activated (RagA/B GTP bound and RagC/D GDP bound) mTORC1
is recruited to the lysosome and activated. Multiple studies have identified addi-
tional Rag GTPase interacting proteins implicated in this pathway (Table 12.1 ).
Amino acids are thought to communicate to the Rag-GTPases, which directly bind
to and somehow redistribute mTORC1 to the surface of lysosomes. Rheb, a potent
mTORC1 activator mediating growth factors, is also thought to reside at the
lysosome. Thus, the lysosome serves as a hub coordinating multiple stimuli to
achieve optimal mTORC1 activation and cell growth.