Toxicity of the Model Protein 3×GFP Arises from Degradation Overload, Not from Aggregate Formation

Although protein aggregation can cause cytotoxicity, such aggregates can also form to mitigate cytotoxicity from misfolded proteins, although the nature of these contrasting aggregates remains unclear. We previously found that overproduction (op) of a three green fluorescent protein-linked protein (3xGFP) induces giant aggregates and is detrimental to growth. Here, we investigated the mechanism of growth inhibition by 3xGFP-op using non-aggregative 3xMOX-op as a control in Saccharomyces cerevisiae. . The 3xGFP aggregates were induced by misfolding, and 3xGFP-op had higher cytotoxicity than 3xMOX-op because it perturbed the ubiquitin-proteasome system. Static aggregates formed by 3xGFP-op dynamically trapped Hsp70 family proteins (Ssa1 and Ssa2 in yeast), causing the heat-shock response. Systematic analysis of mutants deficient in the protein quality control suggested that 3xGFP-op did not cause a critical Hsp70 depletion and aggregation functioned in the direction of mitigating toxicity. Artificial trapping of essential cell cycle regulators into 3xGFP aggregates caused abnormalities in the cell cycle. In conclusion, the formation of the giant 3xGFP 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.