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 multiple fluorescent fusion proteins. Importantly, FPs are originally derived from different organisms from jelly fish to corals and each FP displays 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 what is observed for green FP variants, yemRFP and yFusionRed-tagged polyQ expansions show reduced toxicity. However, polyQ expansions tagged with the bright synthetically engineered ymScarlet displayed severe polyQ toxicity. 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
This resulted in Httex1-yFusionRed toxicity and aggregation, albeit to a lower extent compared to the yeast-optimized msfGFP (ymsfGFP)-tagged counterpart
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 yeast-optimized monomeric variant of superfolder GFP (msfGFP) (Jiang et al, 2017)
Summary
Any reports and responses or comments on the article can be found at the end of the article. In this revised version, we added quantitative growth analysis in liquid cultures to compare toxicity of the different fluorescent fusions. To determine if the absence of toxicity of the yFusionRed-tagged 68Q construct was due to its lower expression, we expressed the fusion from a multicopy vector. This resulted in Httex1-yFusionRed toxicity and aggregation, albeit to a lower extent compared to the ymsfGFP-tagged counterpart.
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