A Reporter System for Cytosolic Protein Aggregates in Yeast

Protein misfolding
and aggregation are linked to neurodegenerative
diseases of mammals and suboptimal protein expression within biotechnology.
Tools for monitoring protein aggregates are therefore useful for studying
disease-related aggregation and for improving soluble protein expression
in heterologous hosts for biotechnology purposes. In this work, we
developed a promoter–reporter system for aggregated protein
on the basis of the yeast native response to misfolded protein. To
this end, we first studied the proteome of yeast in response to the
expression of folded soluble and aggregation-prone protein baits and
identified genes encoding proteins related to protein folding and
the response to heat stress as well as the ubiquitin-proteasome system
that are over-represented in cells expressing an aggregation-prone
protein. From these data, we created and validated promoter–reporter
constructs and further engineered the best performing promoters by
increasing the copy number of upstream activating sequences and optimization
of culture conditions. Our best promoter–reporter has an output
dynamic range of approximately 12-fold upon expression of the aggregation-prone
protein and responded to increasing levels of aggregated protein.
Finally, we demonstrate that the system can discriminate between yeast
cells expressing different prion precursor proteins and select the
cells expressing folded soluble protein from mixed populations. Our
reporter system is thus a simple tool for diagnosing protein aggregates
in living cells and should be applicable for the health and biotechnology
industries.