Journal
TRAFFIC
Volume 10, Issue 8, Pages 1034-1046Publisher
WILEY
DOI: 10.1111/j.1600-0854.2009.00930.x
Keywords
Flim; FRET; GFP; Golgi; small GTPase; tethering factor
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Funding
- Biotechnology and Biological Sciences Research Council
- Science and Technology Facilities Council
- Biotechnology and Biological Sciences Research Council [P20269] Funding Source: researchfish
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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.
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