Ac45 appears as a single low-resolution domain
approximately 30 Å by 20 Å by 30 Å in size
(Fig. 4C, light blue), which is consistent with
only the second luminal domain being in the
complex. Further, although mass spectrometry
detected peptides from uncleaved ATP6AP1/
Ac45, it suggested that the cleaved form is
predominantly present (fig. S12, A and C). The
C-terminal transmembraneahelix of ATP6AP1/
Ac45 is homologous with that of the yeast
V-ATPase subunit Voa1p ( 45 ) and is found in
an equivalent position inside the c ring ( 9 ).
This transmembraneahelix stretches approx-
imately halfway across the thickness of the
lipid bilayer, terminating as a C-terminal tail
that interacts with two of the subunits in the
c ring and subunit d1 before entering the cy-
toplasm (Fig. 4C, light blue). ATP6AP2/PRR is
known to associate with mammalian V-ATPase
( 10 , 11 ) and is essential for biogenesis of active
V-ATPase ( 48 ). The strong density for its trans-
membraneahelix in the map suggests that
every complex includes the subunit (fig. S5C).
Consistent with an emerging role for V-ATPases
in cell signaling ( 49 ), the presence of ATP6AP2/
PRR in the complex has tied V-ATPases to the
renin-angiotensin system for regulation of blood
pressure and electrolyte balance ( 10 , 11 , 13 ), Wnt
signaling ( 14 ), and other pathways ( 12 ). The
gene for ATP6AP2/PRR encodes an N-terminal
extracellular or luminal soluble domain and
a transmembrane anchor (Fig. 4B, top). The
soluble domain increases the angiotensin I–
generating activity of renin and can function
in both a membrane-bound form and, when
released by proteolysis, a soluble form ( 50 , 51 ).
In the structure, the renin-activating domain
of ATP6AP2/PRR is missing (Fig. 4D, yellow).
The transmembrane anchor consists of a long
ahelix and a shorta-helical turn, connected
by an extended linker with N- and C-terminal
tails. The location of ATP6AP2/PRR’strans-
membrane region, captured within the stable
c ring, dictates that, after its incorporation into
the c ring, the protein must remain associated
with the V-ATPase. Mass spectrometry detected
some intact ATP6AP2/PRR in the preparation,
but, consistent with the cryo-EM density, the
cleaved transmembrane region alone was much
more abundant (fig. S12, B and D). In contrast
to the renin-angiotensin system, Wnt signaling
relies on ATP6AP2/PRR remaining membrane-
anchored, with interaction of proteins with the
extracellular or luminal part of ATP6AP2/PRR
leading to signaling in the cytoplasm ( 14 ). The
absenceofthesolubledomainofATP6AP2/
PRR from the structure suggests that ATP6AP2/
PRR’s function in Wnt signaling involves either
a subpopulation of intact ATP6AP2/PRR mole-
culesthatarenotassociatedwithV-ATPaseora
different population of V-ATPase or VOcom-
plexes that contain intact ATP6AP2/PRR.
The structure shows numerous interactions
between ATP6AP1/Ac45, ATP6AP2/PRR, sub-
units d1 and c′′, and multiple c-subunits (Fig. 4,
E and F). Because these proteins are all part
of the rotor subcomplex, the interactions per-
sist during rotation and are also found in
rotationalstates2and3(fig.S13).Inthelumen,
the soluble domain of ATP6AP1/Ac45 interacts
with the N-terminal tail of cleaved ATP6AP2/
PRR, the linker that connects subunit c′′to its
N-terminalahelix in the middle of the c ring,
and the N terminus of subunit c(8)(Fig. 4E).
Near the cytoplasmic surface of the VOregion,
theC-terminaltailofATP6AP1/Ac45andthe
short C-terminalahelix of ATP6AP2/PRR, in-
cluding 12 of the 19 residues in its intracellular
domain ( 14 ), are sandwiched between sub-
units of the c ring and subunit d1 (Fig. 4, C
and D, arrow). VOcomplexes assemble in the
endoplasmic reticulum (ER), with the incor-
poration of subunit d allowing ER release
( 52 , 53 ) and subsequent binding of the V 1
region ( 53 ). Free subunit d in the cytoplasm
( 54 ) does not bind the V 1 complexes that are
preassembled there ( 55 ), which suggests that
the conformation of subunit d changes upon
incorporation into VO,makingitcompetent
to interact with subunit D of V 1 (Fig. 1D). In-
deed, subunit d adopts a more open conforma-
tion when it engages subunit D of the central
rotor in the intact V-ATPase and a more closed
conformation when V 1 is detached and sub-
units d and D are disconnected (fig. S14). The
C-terminal tails of ATP6AP1/Ac45 and ATP6AP2/
PRR produce part of the surface onto which
subunit d1 assembles (Fig. 4, C, D, and F). This
structure explains why mutations in ATP6AP1/
Ac45 and ATP6AP2/PRR are associated with
related disease phenotypes ( 12 , 45 , 46 ). It also
explains why ATP6AP1/Ac45 and ATP6AP2/
PRR appear to work together to allow subunit
d binding, ER release, and subsequent V 1 as-
sembly onto VOin the mammalian V-ATPase
( 9 , 56 ). ATP6AP1/Ac45 and ATP6AP2/PRR
could also be involved in the reverse process,
with conformational changes within them
altering the conformation of subunit d1, dis-
rupting its interaction with subunit D, and
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ACKNOWLEDGMENTS
We thank S. Benlekbir for assistance with cryo-EM data collection;
T. Vasanthakumar for advice on ATPase assays and assistance with
Fig. 1A; and S. Angers, P. Brzezinsky, and members of the Rubinstein
laboratory for discussions.Funding:This work was supported by the
Canadian Institutes of Health Research Grant PJT166152 (J.L.R.), grant
69551-ENABLE from the European Research Council (C.V.R.), and
Wellcome Trust Investigator Award 104633/Z/14/Z (C.V.R.). Y.M.A.
was supported by a Restracomp fellowship and a Canadian Institutes
of Health Research Postdoctoral fellowship. J.L.R. was supported by
the Canada Research Chairs program. Cryo-EM data was collected at
the Toronto High-Resolution High-Throughput cryo-EM facility,
supported by the Canada Foundation for Innovation and Ontario
Research Fund.Author contributions:J.L.R. conceived the project
and experimental approach and supervised the research.
S.A.B. made the SidK expression vector and developed an initial
protein purification strategy. Y.M.A. developed the final protein
purification procedure, prepared cryo-EM specimens, collected
cryo-EM data, calculated the cryo-EM maps, built and refined the
atomic models, and performed biochemical assays. D.W. performedAbbaset al.,Science 367 , 1240–1246 (2020) 13 March 2020 6of7
RESEARCH | REPORT