start-ver=1.4 cd-journal=joma no-vol=137 cd-vols= no-issue=11 article-no= start-page=jcs261977 end-page= dt-received= dt-revised= dt-accepted= dt-pub-year=2024 dt-pub=20240612 dt-online= en-article= kn-article= en-subject= kn-subject= en-title= kn-title=Toxicity of the model protein 3×GFP arises from degradation overload, not from aggregate formation en-subtitle= kn-subtitle= en-abstract= kn-abstract=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 (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 in Saccharomyces cerevisiae. The 3×GFP aggregates were induced by misfolding, and 3×GFP-op had higher cytotoxicity than 3×MOX-op because it perturbed the ubiquitin-proteasome system. Static aggregates formed by 3×GFP-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 3×GFP-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 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. en-copyright= kn-copyright= en-aut-name=NambaShotaro en-aut-sei=Namba en-aut-mei=Shotaro kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=1 ORCID= en-aut-name=MoriyaHisao en-aut-sei=Moriya en-aut-mei=Hisao kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=2 ORCID= affil-num=1 en-affil=Graduate School of Environmental, Life, Natural Science and Technology, Okayama University kn-affil= affil-num=2 en-affil=Faculty of Graduate School of Environmental, Life, Natural Science and Technology, Okayama University kn-affil= en-keyword=Aggregation kn-keyword=Aggregation en-keyword=Fluorescent protein kn-keyword=Fluorescent protein en-keyword=Hsp70 kn-keyword=Hsp70 en-keyword=Overproduction kn-keyword=Overproduction en-keyword=Toxicity kn-keyword=Toxicity en-keyword=Yeast kn-keyword=Yeast END