Toxicity of the model protein 3×GFP arises from degradation overload, not from aggregate formation.

While protein aggregation can cause cytotoxicity, it also forms to mitigate cytotoxicity from misfolded proteins, though the nature of these contrasting aggregates remains unclear. We previously found that overproduction (op) of a three green fluorescent protein linked protein (3×GFP) induces giant aggregates, and is detrimental to growth. Here, we investigated the mechanism of growth inhibition by 3×GFP-op using non-aggregative 3×MOX-op as a control. The 3×GFP aggregates were induced by misfolding, and 3×GFP-op had higher cytotoxicity than 3×MOX-op because it perturbs the ubiquitin-proteasome system. Static aggregates formed by 3×GFP-op dynamically trapped Hsp70, causing the heat shock response. Systematic analysis of mutants deficient in the protein quality control suggested that 3×GFP-op did not cause critical Hsp70 depletion and aggregation functioned in the direction of mitigating toxicity. Artificial trapping of essential cell cycle regulators into 3×GFP aggregates caused abnormalities in the cell cycle. In conclusion, the formation of the giant 3×GFP aggregates itself is not cytotoxic, as it does not entrap and deplete essential proteins. Rather, it is productive, inducing the heat shock response while preventing an overload to the degradation system.