Dosage compensation of Caj1-induced cytotoxicity by Sis1 and Ydj1 reveals complex interactions among JDPs in the yeast cytosol.

Cellular proteostasis depends on tightly regulated Hsp70-J-domain protein (JDP) networks that coordinate protein folding and degradation. Here, we define a previously unrecognized role for the budding yeast JDP Caj1 in modulating nucleocytoplasmic protein quality control. We show that elevated Caj1 levels broadly disrupt proteostasis, stabilizing diverse misfolded substrates by impairing their degradation and triggering the accumulation of ubiquitinated proteins, along with constitutive activation of the heat shock response (HSR). Caj1-induced defects were associated with the accumulation of ubiquitinated proteins. They were genetically suppressed by loss of the E3 ubiquitin ligases and enhanced by loss of deubiquitinating enzymes, revealing functional coupling between Caj1 activity and ubiquitin-mediated protein turnover. Co-overexpression of the major nucleocytoplasmic JDPs Ydj1 and Sis1 suppressed Caj1 toxicity, with Sis1 acting through a J-domain-dependent, Hsp104-independent mechanism. Our findings uncover a dosage-sensitive regulatory interplay among JDPs and demonstrate that relative JDP abundance, rather than absolute levels, is a critical determinant of proteostasis capacity, revealing a previously unrecognized functional network linking Caj1, Sis1, and Ydj1 in shaping cellular protein quality control.