Fluorescence lifetime imaging of interactions between Golgi tethering factors and small GTPases in plants.
Peripheral tethering factors bind to small GTPases in order to obtain their correct location within the Golgi apparatus. Using fluorescence resonance energy transfer (FRET) and fluorescence lifetime imaging microscopy (FLIM) we visualized interactions between Arabidopsis homologues of tethering factors and small GTPases at the Golgi stacks in planta. Co-expression of the coiled-coil proteins AtGRIP and golgin candidate 5 (GC5) [TATA element modulatory factor (TMF)] and the putative post-Golgi tethering factor AtVPS52 fused to green fluorescent protein (GFP) with mRFP (monomeric red fluorescent protein) fusions to the small GTPases AtRab-H1(b), AtRab-H1(c) and AtARL1 resulted in reduced GFP lifetimes compared to the control proteins. Interestingly, we observed differences in GFP quenching between the different protein combinations as well as selective quenching of GFP-AtVPS52-labelled structures. The data presented here indicate that the FRET-FLIM technique should prove invaluable in assessing protein interactions in living plant cells at the organelle level.
Osterrieder
Anne
A
School of Life Sciences, Oxford Brookes University, Oxford, UK.
Carvalho
Claudine M
CM
Latijnhouwers
Maita
M
Johansen
Jorunn Nergaard
JN
Stubbs
Christopher
C
Botchway
Stanley
S
Hawes
Chris
C
D000818:Animals; D017360:Arabidopsis; D029681:Arabidopsis Proteins / Q000235:genetics / Q000378:metabolism*; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D006056:Golgi Apparatus / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D008856:Microscopy, Fluorescence / Q000379:methods*; D020559:Monomeric GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D018515:Plant Leaves / Q000166:cytology / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D053778:Thiazolidines / Q000378:metabolism; D014026:Nicotiana / Q000033:anatomy & histology; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D020798:Two-Hybrid System Techniques
D029681:Arabidopsis Proteins; D019086:Bridged Bicyclo Compounds, Heterocyclic; D004268:DNA-Binding Proteins; C496228:GRIP protein, Arabidopsis; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D053778:Thiazolidines; D014157:Transcription Factors; D049452:Green Fluorescent Proteins; D020559:Monomeric GTP-Binding Proteins; C037068:latrunculin B