Comparison of endogenously expressed fluorescent protein fusions behaviour for protein quality control and cellular ageing research

Abstract

The yeast Hsp104 protein disaggregase is often used as a reporter for misfolded or damaged protein aggregates and protein quality control and ageing research. Observing Hsp104 fusions with fluorescent proteins is a popular approach to follow post stress protein aggregation, inclusion formation and disaggregation. While concerns that bigger protein tags, such as genetically encoded fluorescent tags, may affect protein behaviour and function have been around for quite some time, experimental evidence of how exactly the physiology of the protein of interest is altered within fluorescent protein fusions remains limited. To address this issue, we performed a comparative assessment of endogenously expressed Hsp104 fluorescent fusions function and behaviour. We provide experimental evidence that molecular behaviour may not only be altered by introducing a fluorescent protein tag but also varies depending on such a tag within the fusion. Although our findings are especially applicable to protein quality control and ageing research in yeast, similar effects may play a role in other eukaryotic systems.

The effect of stress on biophysical characteristics of misfolded protein aggregates in living Saccharomyces cerevisiae cells

“…Moreover, a number of optical methods enabling 3D visualisation of individual molecules within living cells have also been developed (reviewed in [46]). However, fluorescent imaging requires appropriate tagging of the molecules of interest which may not only significantly affect their behaviour but also vary depending on a chosen fluorophore [47][48][49]. To overcome these limitations, super-resolution label-free approaches are being developed and successfully applied in studies of complex biological systems [50][51][52][53].…”

The Effect of Lithium on the Budding Yeast Saccharomyces cerevisiae upon Stress Adaptation

“…Over a timespan of 90 min, we followed the formation of aggregates via the GFP-tagged protein disaggregase Hsp104 (hereafter named Hsp104 aggregates) following salt stress. Hsp104 has been used in multiple studies to indicate the frequency and localisation of aggregates in yeast cells due to its ability to bind misfolded proteins [ 30 , 31 ].…”

Boosting the toolbox for live imaging of translation

“…The development of fluorescent proteins is a very rapidly evolving field and FP-based tools need to be updated to optimize their use. Often, the choice of the FP needs to be empirically defined for each cell type, tissue or organism studied [55][56][57][58][59][60] . One important aspect in this choice is the monomeric property of the FP to prevent aggregation issues.…”