TY - JOUR
T1 - Microcompartmentation of cytosolic aldolase by interaction with the actin cytoskeleton in Arabidopsis
AU - Garagounis, Constantine
AU - Kostaki, Kalliopi Ioanna
AU - Hawkins, Tim J.
AU - Cummins, Ian
AU - Fricker, Mark D.
AU - Hussey, Patrick J.
AU - Hetherington, Alistair M.
AU - Sweetlove, Lee J.
PY - 2017/2
Y1 - 2017/2
N2 - 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 noncompetitive 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 actinbinding 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.
AB - 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 noncompetitive 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 actinbinding 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.
KW - Actin-binding
KW - Actin-bundling
KW - Aldolase
KW - Co-localization
KW - Guard cell actin
KW - Guard cell metabolism
KW - Microcompartmentation
UR - http://www.scopus.com/inward/record.url?scp=85018963637&partnerID=8YFLogxK
U2 - 10.1093/jxb/erx015
DO - 10.1093/jxb/erx015
M3 - Article (Academic Journal)
C2 - 28338736
AN - SCOPUS:85018963637
VL - 68
SP - 885
EP - 898
JO - Journal of Experimental Botany
JF - Journal of Experimental Botany
SN - 0022-0957
IS - 5
ER -