During the past decade, it has become clear that protein function and regulation are highly dependent upon intracellular localization. Although fluorescent protein variants are ubiquitously used to monitor protein dynamics, localization and abundance; fluorescent light microscopy techniques often lack the resolution to explore protein heterogeneity and cellular ultrastructure. Several approaches have been developed to identify, characterize and monitor the spatial localization of proteins and complexes at the suborganelle level, yet many of these techniques have not been applied to yeast. Thus, we have constructed a series of cassettes containing codon-optimized epitope tags, fluorescent protein variants that cover the full spectrum of visible light, a TetCys motif used for fluorescein arsenical hairpin (FlAsH)-based localization, and the first evaluation in yeast of a photoswitchable variant, mEos2, to monitor discrete subpopulations of proteins via confocal microscopy. This series of modules, complete with six different selection markers, provides the optimal flexibility during live-cell imaging and multicolour labelling in vivo. Furthermore, high-resolution imaging techniques include the yeast-enhanced TetCys motif, which is compatible with diaminobenzidine photo-oxidation used for protein localization by electron microscopy, and mEos2, which is ideal for super-resolution microscopy. We have examined the utility of our cassettes by analysing all probes fused to the C-terminus of Sec61, a polytopic membrane protein of the endoplasmic reticulum of moderate protein concentration, in order to directly compare fluorescent probes, their utility and technical applications. Our series of cassettes expand the repertoire of molecular tools available to advance targeted spatiotemporal investigations using multiple live-cell, super-resolution or electron microscopy imaging techniques.
Copyright © 2012 John Wiley & Sons, Ltd.