Abstract
Development of fluorescent proteins (FPs) enabled researchers to visualize protein localization and trafficking in living cells and organisms. The extended palette of available FPs allows simultaneous detection of multiples fluorescent fusion proteins. Importantly, FPs are originally derived from different organisms from jelly fish to corals and each FP display its own biophysical properties. Among these properties, the tendency of FPs to oligomerize inherently affects the behavior of its fusion partner. Here we employed the budding yeast Saccharomyces cerevisiae to determine the impact of the latest generation of red FPs on their binding partner. We used a yeast assay based on the aggregation and toxicity of misfolded polyQ expansion proteins linked to Huntington’s disease. Since polyQ aggregation and toxicity are highly dependent on the sequences flanking the polyQ region, polyQ expansions provide an ideal tool to assess the impact of FPs on their fusion partners. We found that unlike yemRFP and yFusionRed, the synthetically engineered ymScarlet displayed severe polyQ toxicity and aggregation similar to what is observed for green FP variants. Our data indicate that ymScarlet might have significant advantages over the previous generation of red FPs for use in fluorescent fusions in yeast.
Highlights
Following the development of the green fluorescent protein (GFP) from the jellyfish Aqueaora victoria (Chalfie et al, 1994), several other fluorescent proteins (FPs) with various spectral properties have been characterized (Thorn, 2017), allowing simultaneous detection of multiple fluorescent reporters
Using polyQ toxicity assays in yeast, we previously showed that a yeast-optimized version of mCherry (termed yemRFP (Keppler-Ross et al, 2008)) displays only a mild growth defects compared to yeastoptimized monomeric variant of superfolder GFP (msfGFP) (Jiang et al, 2017)
DNA constructs yemRFP (Keppler-Ross et al, 2008) was previously described. yFusionRed and ymScarlet were codon optimized for expression in yeast and synthetized by Genscript Inc. based on previously published sequences (Bindels et al, 2017; Shemiakina et al, 2012)
Summary
Following the development of the green fluorescent protein (GFP) from the jellyfish Aqueaora victoria (Chalfie et al, 1994), several other FPs with various spectral properties have been characterized (Thorn, 2017), allowing simultaneous detection of multiple fluorescent reporters. Using polyQ toxicity assays in yeast, we previously showed that a yeast-optimized version of mCherry (termed yemRFP (Keppler-Ross et al, 2008)) displays only a mild growth defects compared to yeastoptimized msfGFP (ymsfGFP) (Jiang et al, 2017). These results lead us to exploit the polyQ toxicity and aggregation assays to explore to effects of two of the most recently available RFPs. Here, we focused on FusionRed, a red monomeric fluorescent variant of mKate2 known for its low cytotoxicity in cells (Shemiakina et al, 2012) that displays low propensity to oligomerize in mammalian cells (Costantini et al, 2015).
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