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

Harvard University, Cambridge, Massachusetts, United States
AJP Cell Physiology (Impact Factor: 3.78). 02/2011; 300(6):C1442-55. DOI: 10.1152/ajpcell.00076.2010
Source: PubMed


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.

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    • "The V-ATPase is a complex enzyme, composed of at least 14 subunits assembled into a transmembrane, proton- (H+-) translocating V0 sector and a cytosolic, catalytic V1 domain (Fig. 1) [55], [56], [57], [58]. In mammalian tissues the 56-kDa B subunit of the enzyme is expressed as two isoforms, B1 and B2. "
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    • "V-ATPases have also been shown to interact with several glycolytic enzymes [15-19], which are known to also bind microfilaments [20-24]. Recently, interactions between V-ATPases, fructose bisphosphate aldolase and ARNO were described which may signify the emergence of a mechanism by which the spatial localization and activity of V-ATPases are coupled to the metabolic state of the cell [11]. "
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