Aldolase directly interacts with ARNO and modulates cell morphology and acidic vesicle distribution

Abstract

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 interaction 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: K(D) = 2.84 × 10(-10) M. MTC cell fractionation revealed that aldolase is also associated with membranes of early endosomes. Functionally, aldolase knockdown in HeLa cells produced striking morphological changes accompanied by long filamentous cell protrusions and acidic vesicle redistribution. However, the 50% knockdown we achieved did not modulate the acidification capacity of endosomal/lysosomal compartments. Finally, a combination of small interfering RNA knockdown and overexpression revealed 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 compartments via a combination of protein-protein interaction and gene expression mechanisms.

Subunit Interactions at the V1-Vo Interface in Yeast Vacuolar ATPase

“…In addition, subunit a has been proposed at act as a pH sensor (54) by sensing lumenal pH with its membrane integral C-terminal domain and transmitting this information to cellular binding partners via the soluble a NT . These findings are on the basis of the intraendosomal pH dependence of the interaction of subunit a NT with the ADP-ribosylation factor guanine nucleotide exchange factor (Arf-GEF), ADP ribosylation factor nucleotide site opener (55), and the glycolytic enzyme aldolase (56).…”

The vacuolar-type H+-ATPase at a glance – more than a proton pump

“…Aldolase interacts with the VATPase subunits a, B and E, and this interaction is required to stabilize the assembled V-ATPase on the membrane (Lu et al, 2007;Lu et al, 2001;Lu et al, 2004;Merkulova et al, 2010;Merkulova et al, 2011). Indeed, aldolase separates from the V-ATPase upon glucose starvation , underlying the reported dissociation of the V 1 and V 0 domains in a microtubuledependent manner (Tabke et al, 2014;Xu and Forgac, 2001).…”

Saccharomyces cerevisiae Vacuolar H + -ATPase Regulation by Disassembly and Reassembly: One Structure and Multiple Signals

“…The interrelation between V-ATPase and glycolysis cannot be overlooked; it is conserved in yeast (1,19,35,36), plants (37), and mammals (38,39). Moreover, V-ATPase mutations that impair binding to phosphofructokinase-1 are associated with distal renal tubular acidosis (24), and V-ATPase regulation by glycolysis plays a role in viral infections (40) and the metabolic switch in cancers (41,42).…”