Microcompartmentation of plant glycolytic enzymes with subcellular structures

Please use this identifier to cite or link to this item:
https://osnadocs.ub.uni-osnabrueck.de/handle/urn:nbn:de:gbv:700-2009102118
Open Access logo originally created by the Public Library of Science (PLoS)
Title: Microcompartmentation of plant glycolytic enzymes with subcellular structures
Authors: Wojtera, Joanna
Thesis advisor: Prof. Dr. Renate Scheibe
Thesis referee: apl. Prof. Dr. Hans Merzendorfer
Abstract: Classically considered as a soluble system of enzymes, glycolysis does not conform to the known function and subcellular microcompartmentation pattern. Certain glycolytic enzymes, such as glyceraldehyde-3-phosphate dehydrogenase (GAPDH) could be found at different cellular locations in animal cells, where it exhibited its non-glycolytic activities. Determination of the subcellular localization of two cytosolic GAPDH isoforms from Arabidopsis thaliana (GapC1 and GapC2), fused to Fluorescent Proteins (FP), was investigated in the transiently transformed mesophyll protoplasts, using confocal Laser Scanning Microscopy. Apart from its cytosolic distribution, the nuclear compartmentation of GapC:FP was observed in this study, as well as its punctuate or aggregate-like localization. Part of the GapC:FP foci were observed as mitochondria-associated. A further yeast two-hybrid screen with both GapC isoforms as baits allowed to identify the mitochondrial porin (VDAC3; At5g15090) as a protein-protein interaction partner. Further tests with one-on-one yeast two-hybrid and Bimolecular Fluorescence Complementation (BiFC) assays showed that the detected binding between plant VDAC3 and GapC in yeast cells was false positive. Interestingly, aldolase interacted with VDAC3, as well as with GapC in plant protoplasts, using the BiFC method. On the other hand, no such interaction could be detected in the one-on-one yeast two-hybrid assay. Thus, a model of indirect mitochondrial association of GapC via aldolase that binds directly to mitochondrial porin is proposed to occur only upon certain cellular conditions. Attempts to show the binding of Arabidopsis GAPDH to the actin cytoskeleton in vivo failed, whereas in vitro cosedimentation assays demonstrated that the fully active, recombinant glycolytic enzyme binds to rabbit F-actin. Moreover, is the presence of the GapC cofactor NAD and a reducing agent (DTT), the enzyme might exhibit an actin-bundling activity.
URL: https://repositorium.ub.uni-osnabrueck.de/handle/urn:nbn:de:gbv:700-2009102118
Subject Keywords: glycolysis; GAPDH; aldolase; subcellular microcompartmentation; protein-protein interaction; mitochondrial porin; actin cytoskeleton
Issue Date: 20-Oct-2009
Type of publication: Dissertation oder Habilitation [doctoralThesis]
Appears in Collections:FB05 - E-Dissertationen

Files in This Item:
File Description SizeFormat 
E-Diss951_thesis.pdfPräsentationsformat11,1 MBAdobe PDF
E-Diss951_thesis.pdf
Thumbnail
View/Open
E-Diss951_supplement.tar.gz13,34 MBGZIP
E-Diss951_supplement.tar.gz
View/Open


Items in osnaDocs repository are protected by copyright, with all rights reserved, unless otherwise indicated. rightsstatements.org