Aldolase directly interacts with ARNO and modulates cell morphology
and acidic vesicle distribution
Maria Merkulova, Andrés Hurtado-Lorenzo, Hiroyuki Hosokawa, Zhenjie Zhuang, Dennis Brown,
Dennis A. Ausiello, and Vladimir Marshansky
Program in Membrane Biology and Nephrology Division, Center for Systems Biology, Simches Research Center,
Massachusetts General Hospital and Department of Medicine, Harvard Medical School, Boston, Massachusetts
Submitted 8 March 2010; accepted in final form 2 February 2011
Merkulova M, Hurtado-Lorenzo A, Hosokawa H, Zhuang Z,
Brown D, Ausiello DA, Marshansky V. Aldolase directly interacts
with ARNO and modulates cell morphology and acidic vesicle dis-
tribution. Am J Physiol Cell Physiol 300: C1442–C1455, 2011. First
published February 9, 2011; doi:10.1152/ajpcell.00076.2010.—
Previously, we demonstrated that the vacuolar-type H?-ATPase
(V-ATPase) a2-subunit functions as an endosomal pH sensor that
interacts with the ADP-ribosylation factor (Arf) guanine nucleotide
exchange factor, ARNO. In the present study, we showed that ARNO
directly interacts not only with the a2-subunit but with all a-isoforms
(a1–a4) of the V-ATPase, indicating a widespread regulatory interac-
tion between V-ATPase and Arf GTPases. We then extended our
search for other ARNO effectors that may modulate V-
ATPase-dependent vesicular trafficking events and actin cytoskeleton
remodeling. Pull-down experiments using cytosol of mouse proximal
tubule cells (MTCs) showed that ARNO interacts with aldolase, but
not with other enzymes of the glycolytic pathway. Direct interaction
of aldolase with the pleckstrin homology domain of ARNO was
revealed by pull-down assays using recombinant proteins, and surface
plasmon resonance revealed their high avidity interaction with a
dissociation constant: KD? 2.84 ? 10?10M. MTC cell fractionation
revealed that aldolase is also associated with membranes of early
endosomes. Functionally, aldolase knockdown in HeLa cells pro-
duced striking morphological changes accompanied by long filamen-
tous cell protrusions and acidic vesicle redistribution. However, the
50% knockdown we achieved did not modulate the acidification
capacity of endosomal/lysosomal compartments. Finally, a combina-
tion of small interfering RNA knockdown and overexpression re-
vealed that the expression of aldolase is inversely correlated with
gelsolin levels in HeLa cells. In summary, we have shown that
aldolase forms a complex with ARNO/Arf6 and the V-ATPase and
that it may contribute to remodeling of the actin cytoskeleton and/or
the trafficking and redistribution of V-ATPase-dependent acidic com-
partments via a combination of protein-protein interaction and gene
ADP-ribosylation factor nucleotide site opener; vacuolar H?-ATPase;
a-subunit isoforms; actin cytoskeleton; gelsolin; surface plasmon
resonance; endosomal/lysosomal compartments
FRUCTOSE BISPHOSPHATE ALDOLASE (EC 184.108.40.206) is the fourth
enzyme of glycolysis, which catalyses reversible cleavage of
fructose 1,6-bisphosphate into glyceraldehyde 3-phosphate and
dihydroxyacetone phosphate. Aldolase has been studied for
over 70 years and is a very well-characterized protein. The
success in studying this protein is in part due to its high
abundance and relatively easy purification from natural sources.
Several crystal structures of wild-type and mutant proteins are
available now, which clarify the mechanism of its catalytic
activity (2, 17, 18, 38). Although the role of aldolase in
carbohydrate metabolism is well established, there is growing
evidence for many alternative so-called “moonlighting” func-
tions for this enzyme. In particular, aldolase interacts with
various proteins unrelated to glycolytic enzymes, including
F-actin, ?-tubulin, dynein light chain 8, the actin nucleation
promoting factor WASP, the endocytotic sorting protein
nexin-9, and phospholipase D2(10, 34, 55, 71, 73, 74). These
multiple interactions indicate that aldolase is involved, proba-
bly as a scaffolder, in the coordination of membrane trafficking
and cytoskeleton dynamics at the cell periphery. As a result, a
novel emerging field for aldolase biology is its central role in
a variety of vesicular trafficking events, including 1) endocy-
tosis and parasite invasion, 2) cytoskeleton rearrangement and
cell motility, 3) trafficking and recycling of membrane pro-
teins, and 4) signal transduction.
Interaction of aldolase with vacuolar-type H?-ATPase
(V-ATPase) was also reported previously (6, 35–37, 58). The
V-ATPase is a multimeric proton-pumping protein complex
that is found on plasma membranes and diverse endomembrane
organelles. The biochemistry, cell biology, and pathophysiol-
ogy of V-ATPase have been extensively reviewed by ourselves
and others (9, 24, 42, 47, 72). The cytoplasmic V1sector of the
V-ATPase is composed of eight different subunits with defined
stoichiometry (A3B3CDE3FG3H) and is responsible for ATP
hydrolysis (20, 45, 75). The transmembrane V0 sector is
composed of six different subunits (ac5c==deAc45) and is
responsible for proton translocation across the lipid bilayer.
Consistent with the presence of V-ATPases in diverse subcel-
lular compartments, a large spectrum of subunit isoforms and
potential splice variants have been identified (9, 24, 42, 44, 47,
64, 72). The expression of these isoforms is tissue and cell
specific. Intracellular targeting and assembly of V-ATPase in
the functional holocomplex is regulated by a-subunit isoforms,
four of which (a1, a2, a3, and a4) are found in mice and
humans (49, 63, 65, 69). V-ATPase activity is controlled by
different mechanisms, including the physical disassembly of
V1 and V0 sectors of V-ATPase (31, 50). Disassembled
V-ATPase is no longer able to hydrolyze ATP, and thus this
nanomotor cannot pump protons across the membranes. Aldo-
lase interacts with three different subunits of the V-ATPase:
the transmembrane a-subunit of the V0sector, as well as the
soluble E- and B-subunits of the V1sector (36, 37). In yeast
and mammalian kidney cells, the interaction between aldolase
and the V-ATPase is modified by glucose, suggesting that
aldolase may act as a glucose sensor and mediate V-ATPase
Address for reprint requests and other correspondence: V. Marshansky,
Program in Membrane Biology and Nephrology Division, Center for Systems
Biology, Simches Research Center, Massachusetts General Hospital, 185 Cam-
bridge St., CPZN, Suite 8212, Boston, MA 02114 (e-mail: Vladimir_Marshansky
Am J Physiol Cell Physiol 300: C1442–C1455, 2011.
First published February 9, 2011; doi:10.1152/ajpcell.00076.2010.
0363-6143/11 Copyright © 2011 the American Physiological Societyhttp://www.ajpcell.org C1442
assembly/disassembly and, therefore, its function. However,
further analysis demonstrated that aldolase enzymatic activity
is not required for regulation of V-ATPase assembly/disassem-
bly, suggesting an important role of physical association in
the V-ATPase/aldolase complex (35). On the other hand,
V-ATPase assembly/disassembly in yeast is also modulated by
the Ras/cAMP/PKA pathway (6). In particular, upregulation of
the Ras/cAMP/PKA pathway by expression of constitutively
active PKA blocks glucose-dependent V-ATPase dissociation.
Moreover, overexpression of Ras leads to decreased binding of
aldolase to V-ATPase (6).
The Ras-superfamily small GTPases, which includes ADP-
ribosylation factor small GTPase (Arf) proteins, function as
“molecular switches” and regulate an extraordinary variety of
cell functions (7). The transition between “on” and “off” states
of this molecular device is mediated by a GDP/GTP cycle. Six
Arfs have been identified in mammalian cells, and they are all
critical for the regulation of vesicular trafficking both in exo-
and endocytotic pathways (16, 21, 22). Whereas Arf1 is in-
volved in regulating endoplasmic reticulum (ER)-Golgi traf-
ficking (14, 60), Arf6 is targeted to the plasma membrane
and/or endosomes and is involved in the regulation of endo-
cytosis and membrane recycling (3, 46, 59). Activation of Arfs
is achieved by GDP/GTP exchange in the presence of guanine
nucleotide exchange factors (GEFs). ADP-ribosylation factor
nucleotide site opener (ARNO) is one of four members of the
cytohesin subfamily of Arf-GEFs, all of which share the
following structural domains: 1) an NH2-terminal coiled-coil
(CC), 2) a central Sec7 domain, 3) a pleckstrin homology (PH)
domain, and 4) a COOH-terminal polybasic (PB) domain (11,
21, 30). Functionally, both Arf6 and ARNO have been
implicated in regulation of the endocytotic pathway, organ-
elle biogenesis, and actin cytoskeleton remodeling (11, 16,
21, 22, 56).
Recent work from our laboratory has uncovered a crucial
link between V-ATPase and Arf-family small GTPases in
regulation of the endosomal/lysosomal protein degradative
pathway (29, 41). We demonstrated that V-ATPase has a novel
function as endosomal pH sensor that scaffolds ARNO and
Arf6. The transmembrane a2-subunit of the V-ATPase directly
interacts with cytosolic ARNO, whereas the V-ATPase c-sub-
unit specifically binds Arf6; these biochemical events are
essential for vesicular trafficking between early and late endo-
somal compartments (41). However, the molecular mechanism
and cell biological significance of the interactions between
V-ATPase and small GTPases remain obscure. Recently, we
mapped the interaction sites between the NH2-terminal tail of
the V-ATPase a2-subunit (a2N) and ARNO and demonstrated
the crucial role of the catalytic Sec7 domain in this interaction
(43). Moreover, we also recently discovered a novel function
of the V-ATPase as a regulator of the GEF activity of ARNO
and, thus, as a modulator of Arf-small GTPase signaling (28).
Although this previous work uncovered a functional cross
talk between V-ATPase, ARNO, and Arf small GTPases, other
downstream effectors and related cell biological events have
also been unraveled. In the present study, we hypothesize that
since V-ATPase interacts with both ARNO (29) and aldolase
(35), these two proteins could in turn interact with each other
and coordinate endocytic vesicle trafficking and cytoskeleton
rearrangements. To uncover the downstream effectors of
ARNO and V-ATPase signaling, we have now applied a
combination of protein-protein interaction techniques, and we
indeed identify aldolase as a specific and high-affinity interac-
tion partner of ARNO that could be involved in intracellular
trafficking and cytoskeletal modulation.
MATERIALS AND METHODS
Reagents and antibodies. If not otherwise specified, all reagents,
including D(?)-glucose, bafilomycin A1, aldolase type IV from rabbit
muscle (aldolase-A), bovine albumin-FITC, and iodixanol (OptiPrep
density gradient medium) were obtained from Sigma (St. Louis, MO).
Lipofectamine 2000 transfection reagent, Alexa Fluor 647 phalloidin,
fluorescein-dextran (10,000 MW, anionic), and the acidic organelle
probes DAMP and LysoTracker red DND-99 were obtained from
Invitrogen (Carlsbad, CA). NuPAGE gels and buffers were also
acquired from Invitrogen. EDTA-free Complete protease inhibitor
tablets were obtained from Roche (Indianapolis, IN). Octylglucoside
(n-octyl-?-D-glucopyranoside) was obtained from Anatrace (Maumee,
OH). Western Lightning chemiluminescence reagent plus was ob-
tained from PerkinElmer (Boston, MA). TALON metal affinity resin
was acquired from Clontech (Mountain View, CA), and glutathione-
Sepharose 4B beads were obtained from GE Healthcare (Piscataway,
NJ). Metabolic labeling of recombinant proteins during in vitro
translation was achieved by incorporation of either [35S]methionine
purchased from GE Healthcare or BODIPY-lysine-tRNA purchased
from Promega (Madison, WI). Thrombin cleavage capture kit was
purchased from EMD-Biosciences/Novagen (Gibbstown, NJ). Bicin-
choninic acid (BCA) protein assay kit was obtained from Thermo
Fisher Scientific (Rockford, IL). Affinity-purified rabbit polyclonal
anti-aldolase-B antibodies (HPA002198) and mouse monoclonal anti-
gelsolin antibodies (GS-2C4) were purchased from Sigma. Affinity-
purified goat polyclonal anti-actin (C-11) antibodies and affinity-
purified goat polyclonal anti-aldolase-A/B (D-18) antibodies were
purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The
following affinity-purified antibodies against glycolytic enzymes were
also purchased from Santa Cruz Biotechnology: goat polyclonal
anti-glyceraldehyde-3-phosphate dehydrogenase (anti-GAPDH; V-18), goat
polyclonal anti-phosphofructokinase (anti-PFK; E-16), goat poly-
clonal anti-phosphoglycerate kinase (anti-PGK; E-20), and rabbit
polyclonal anti-enolase (H-300). Rabbit polyclonal anti-FITC (A-889)
and Alexa 488-conjugated anti-dinitrophenyl-KLH (anti-DNP) anti-
bodies (A-11097) were obtained from Invitrogen. Mouse monoclonal
anti-megalin (1H2) antibodies were previously described (26, 29).
The following secondary antibodies were also used: goat anti-rabbit
IgG-horseradish peroxidase (RPN4301) and sheep anti-mouse IgG-
horseradish peroxidase (NA931V) were purchased from GE Health-
care, and donkey anti-goat IgG-horseradish peroxidase (sc-2020) was
purchased from Santa Cruz Biotechnology.
DNA constructs, recombinant protein expression, and purification.
cDNAs encoding full-length wild-type mouse V-ATPase a1-isoform
(832 aa, GI no. 7363245), a2-isoform (856 aa, GI no. 7363249),
a3-isoform (834 aa, GI no. 7363247), and a4-isoform (833 aa, GI no.
13990958) were generously provided by Dr. Masamitsu Futai (Iwate
Medical University, Iwate, Japan). cDNA constructs encoding the
NH2-terminal cytosolic tail of mouse V-ATPase a-isoforms a1N
(1–388 aa), a2N (1–393 aa), a3N (1–386 aa), and a4N (1–390 aa) were
amplified using the Expand High Fidelity PCR system and subcloned
into a pIVEX2.4d vector (Roche) as previously described (29). The
resulting constructs contain a modified NH2-terminal 6XHis-tag
(MSGSHHHHHHSSGIEGRGRLIKMT). All four constructs were in
vitro translated and metabolically labeled with [35S]methionine using
the RTS100 kit (Roche). For in vitro translation and pull-down
experiments with aldolase, the human aldolase-B cDNA was obtained
from ATCC (no. MGC-32618, I.M.A.G.E. clone ID 4593670, Gen-
Bank accession nos. BC029399, BG402660). This cDNA was cloned
into pIVEX2.3 vector (Roche). This construct was in vitro translated
using the RTS100 kit (Roche) with or without labeling by FluoroTect
ALDOLASE INTERACTS WITH ARNO AND MODULATES CELL MORPHOLOGY
AJP-Cell Physiol • VOL 300 • JUNE 2011 • www.ajpcell.org
V-ATPase. Thus these data suggest that the interaction of
ARNO with aldolase and V-ATPases, which are specifically
targeted to different subcellular compartments by the a-subunit
isoforms, is a widespread phenomenon that may take place on
a variety of cell membranes and organelles. In searching for the
functional significance of this interaction, we uncovered a
critical role of aldolase in actin cytoskeleton remodeling that
results in the formation of cell protrusions and acidic vesicle
redistribution. Thus we propose that aldolase is a novel mod-
ulator of V-ATPase/Arf6/ARNO and actin/gelsolin signaling
pathways and is involved in the coordination of actin-depen-
dent cell shape remodeling and acidic vesicle redistribution.
However, the detailed interplay between these pathways and
the intertwined role of aldolase in regulation of both vesicular
trafficking and the cellular cytoskeleton remain to be tested in
We thank Dr. Masamitsu Futai for providing cDNAs of the full-length
wild-type mouse V-ATPase a1-, a2-, a3-, and a4-isoforms. We are also grateful
to Dr. Jim Casanova and Dr. Sylvain Bourgoin for providing cDNA of
triglycine variant of human wild-type ARNO. We thank Dr. Dean Tolan and
Carolyn Ritterson Lew (Boston University) for providing reagents, equipment,
and training to perform aldolase activity assay and also for constructive
suggestions and discussions during preparation of aldolase-A siRNA knock-
This work was supported by National Institute of Diabetes and Digestive
and Kidney Diseases (NIDDK) Grant DK038452 (to V. Marshansky, D.
Brown, and D. A. Ausiello) and a pilot and feasibility study supported by a
Boston Area Diabetes Research Center NIDDK Grant DK057521-08 (to V.
No conflicts of interest, financial or otherwise, are declared by the author(s).
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ALDOLASE INTERACTS WITH ARNO AND MODULATES CELL MORPHOLOGY
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