Mixed‐class J‐domain protein scaffolds promote expanded aggregate handling and multivalent Hsp70 engagement during functional disaggregase assembly

Protein aggregates threaten cellular proteostasis and are linked to aging and disease. In metazoa, aggregate resolution relies on Hsp70‐J‐domain protein (JDP)‐based disaggregases. Previous studies showed human class A and class B JDP assemblies enhance Hsp70‐mediated disaggregation, but the underlying mechanism has remained unclear. Using J‐domain mutants that impair Hsp70 binding while preserving mixed‐class JDP interaction, we show that synergistic disaggregation is lost when either JDP partner cannot engage Hsp70. Size‐resolved disaggregation assays further reveal that mixed‐class JDP assemblies influence the processing of distinct luciferase aggregate populations, including aggregate species inefficiently handled by either JDP alone. Our findings support a model in which mixed‐class JDP assemblies enhance Hsp70 disaggregation through expanded aggregate‐processing capacity and multivalent Hsp70 recruitment by both JDP partners.