Polyglutamine toxicity in yeast uncovers phenotypic variations between different fluorescent protein fusions

Abstract

The palette of fluorescent proteins (FPs) available for live-cell imaging contains proteins that strongly differ in their biophysical properties. FPs cannot be assumed to be equivalent and in certain cases could significantly perturb the behavior of fluorescent reporters. We employed Saccharomyces cerevisiae to comprehensively study the impact of FPs on the toxicity of polyglutamine (polyQ) expansion proteins associated with Huntington's disease. The toxicity of polyQ fusion constructs is highly dependent on the sequences flanking the polyQ repeats. Thus, they represent a powerful tool to study the impact of fluorescent fusion partners. We observed significant differences on polyQ aggregation and toxicity between commonly used FPs. We gener-

Sfp1 regulates the SAGA component Tra1 in response to proteotoxic stress inSaccharomyces cerevisiae

“…SPT7 promoter sequences relative to the translational start, -633 to +68, NGG1 promoter sequences -430 to +5 and EAF1 promoter sequences -890 to +31 were engineered by PCR as BamHI/HindIII fragments using oligonucleotides listed in Table 3 and cloned into YCplac87 60 to generate transcriptional reporters. Vectors encoding fluorescently tagged Tra1 with either ysmfGFP 52 and yemRFP 61 were generated by replacing the eGFP coding sequence by the new codon-optimized fluorescent proteins using the BamHI/NotI sites in the previously described eGFP-Tra1 vector 62 using primers listed in Supplemental Table 3 .…”

“…To study the effect of Htt ex1 polyQ expansion on yeast, we employed a well-characterized model that involves expressing fluorescently-tagged Htt ex1 48–52 . We define the interplay between Tra1 and polyQ-induced stress.…”

Studying Huntington’s Disease in Yeast: From Mechanisms to Pharmacological Approaches

“…However, this early model did not show cytotoxicity. Later studies revealed a strong dependency of mHTT toxicity on the flanking sequences ( Dehay and Bertolotti, 2006 ; Duennwald et al, 2006a ; Jiang et al, 2016 ), which might explain the lack of cytotoxicity in the model used by Krobitsch and Lindquist (2000) . Interestingly, shortly after this study, it was shown that in a similar model redirecting fragmented exon-1-poly(Q)-fusion proteins to the nucleus, nuclear aggregates are formed, which are unaffected by HSP104 deletion ( Cao et al, 2001 ).…”

“…Importantly, yeast-optimized C-terminal fluorescent proteins (FPs) also affect phenotypes caused by exon1-Htt103Q expression ( Jiang et al, 2016 ). The presence of an FP was essential for elongated poly(Q) phenotypes per se , and both aggregation patterns and toxicity were altered by different FPs.…”

“…The presence of an FP was essential for elongated poly(Q) phenotypes per se , and both aggregation patterns and toxicity were altered by different FPs. From the tested FPs, the blue FP yomTagBFP2 prevented Htt72Q aggregation and toxicity, probably via its strong tendency to self-assemble into oligomers, which could prevent mHTT-specific inclusion body formation ( Jiang et al, 2016 ). Altogether, flanking regions can profoundly modulate cytotoxicity in yeast and other systems ( Lakhani et al, 2010 ).…”