Spatiotemporal dynamics of Pma1 in Saccharomyces cerevisiae using a reversibly interacting tag-probe system
Abstract
When fluorescent proteins (FPs) are directly fused to a protein of interest, they can interfere with wild-type protein functionality. For example, when plasma membrane (PM) protein Pma1, in Saccharomyces cerevisiae, is directly fused to an FP, it is mislocalized to the cells' vacuoles. To circumvent such problems, we are developing a versatile live-cell imaging strategy in which a fluorescently-labeled tetratricopeptide repeat affinity protein (TRAP) probe can reversibly bind to a conjugate 5 amino-acid peptide tag, which is bound to the target protein's C-terminus. We demonstrate the utility of this reversibly binding tag-probe system, showing that it properly localizes Pma1 to the PM. To further characterize the behavior of Pma1, we present the results of fluorescence recovery after photobleaching (FRAP), and single-particle tracking photoactivated localization microscopy (sptPALM) measurements under total internal reflection fluorescence (TIRF) illumination. Remarkably, we find significant differences in the diffusional dynamics of Pma1 imaged using our novel labeling methodology, and Pma1 directly fused to FP.
We acknowledge support of the NIGMS via AWDA10958.- Publication:
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APS March Meeting Abstracts
- Pub Date:
- 2019
- Bibcode:
- 2019APS..MARC63013P