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

Am J Physiol Cell Physiol. 2011 Jun;300(6):C1442-55. doi: 10.1152/ajpcell.00076.2010. Epub 2011 Feb 9.

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.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • ADP-Ribosylation Factor 6
  • ADP-Ribosylation Factors / genetics
  • ADP-Ribosylation Factors / metabolism
  • Animals
  • Cell Shape*
  • Cell Surface Extensions / metabolism
  • Cell Surface Extensions / ultrastructure
  • Cytoplasmic Vesicles / metabolism*
  • Endosomes / metabolism
  • Fructose-Bisphosphate Aldolase / genetics
  • Fructose-Bisphosphate Aldolase / metabolism*
  • GTPase-Activating Proteins / genetics
  • GTPase-Activating Proteins / metabolism*
  • Gelsolin / genetics
  • Gelsolin / metabolism
  • Gene Knockdown Techniques
  • HeLa Cells
  • Humans
  • Hydrogen-Ion Concentration
  • Isoenzymes / genetics
  • Isoenzymes / metabolism
  • Mice
  • Protein Subunits / chemistry
  • Protein Subunits / genetics
  • Protein Subunits / metabolism
  • RNA, Small Interfering / genetics
  • RNA, Small Interfering / metabolism
  • Vacuolar Proton-Translocating ATPases / chemistry
  • Vacuolar Proton-Translocating ATPases / genetics
  • Vacuolar Proton-Translocating ATPases / metabolism

Substances

  • ADP-Ribosylation Factor 6
  • GTPase-Activating Proteins
  • Gelsolin
  • Isoenzymes
  • Protein Subunits
  • RNA, Small Interfering
  • cytohesin-2
  • Vacuolar Proton-Translocating ATPases
  • ADP-Ribosylation Factors
  • ARF6 protein, human
  • Arf6 protein, mouse
  • Fructose-Bisphosphate Aldolase