Microcompartmentation of cytosolic aldolase by interaction with the actin cytoskeleton in Arabidopsis.
Evidence is accumulating for molecular microcompartments formed when proteins interact in localized domains with the cytoskeleton, organelle surfaces, and intracellular membranes. To understand the potential functional significance of protein microcompartmentation in plants, we studied the interaction of the glycolytic enzyme fructose bisphosphate aldolase with actin in Arabidopsis thaliana. Homology modelling of a major cytosolic isozyme of aldolase, FBA8, suggested that the tetrameric holoenzyme has two actin binding sites and could therefore act as an actin-bundling protein, as was reported for animal aldolases. This was confirmed by in vitro measurements of an increase in viscosity of F-actin polymerized in the presence of recombinant FBA8. Simultaneously, interaction with F-actin caused non-competitive inhibition of aldolase activity. We did not detect co-localization of an FBA8-RFP fusion protein, expressed in an fba8-knockout background, with the actin cytoskeleton using confocal laser-scanning microscopy. However, we did find evidence for a low level of interaction using FRET-FLIM analysis of FBA8-RFP co-expressed with the actin-binding protein GFP-Lifeact. Furthermore, knockout of FBA8 caused minor alterations of guard cell actin cytoskeleton morphology and resulted in a reduced rate of stomatal closure in response to decreased humidity. We conclude that cytosolic aldolase can be microcompartmented in vivo by interaction with the actin cytoskeleton and may subtly modulate guard cell behaviour as a result.
Department of Plant Sciences, University of Oxford, South Parks Road, Oxford OX1 3RB, UK.
Constantine
C
Garagounis
School of Biological Sciences, University of Bristol, Life Sciences Building, 24 Tyndall Avenue, Bristol BS8 1TQ, UK.
Kalliopi-Ioanna
KI
Kostaki
School of Biosciences, Durham University, South Road, Durham DH1 3LE, UK.
Tim J
TJ
Hawkins
School of Biosciences, Durham University, South Road, Durham DH1 3LE, UK.
Ian
I
Cummins
Department of Plant Sciences, University of Oxford, South Parks Road, Oxford OX1 3RB, UK.
Mark D
MD
Fricker
School of Biosciences, Durham University, South Road, Durham DH1 3LE, UK.
Patrick J
PJ
Hussey
School of Biological Sciences, University of Bristol, Life Sciences Building, 24 Tyndall Avenue, Bristol BS8 1TQ, UK.
Alistair M
AM
Hetherington
Department of Plant Sciences, University of Oxford, South Parks Road, Oxford OX1 3RB, UK.
Lee J
LJ
Sweetlove
D008841:Actin Cytoskeleton / Q000378:metabolism*; D000199:Actins / Q000378:metabolism*; D017360:Arabidopsis / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D005634:Fructose-Bisphosphate Aldolase / Q000235:genetics* / Q000378:metabolism; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D018613:Microscopy, Confocal; D010940:Plant Proteins / Q000235:genetics* / Q000378:metabolism
D000199:Actins; D007527:Isoenzymes; D010940:Plant Proteins; D005634:Fructose-Bisphosphate Aldolase
Actin-binding; actin-bundling; aldolase; co-localization; guard cell actin; guard cell metabolism; microcompartmentation.