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This lecture delves into the genetic exploration of J-domain protein specificity, focusing on the role of Hsp70 and its cochaperones in protein homeostasis, sequestration, and degradation under stress conditions. The essentiality of Sis1, a J-domain protein, is highlighted through its unique functions in protein folding, translocation into mitochondria, and disaggregation of amorphous aggregates. The lecture also discusses the critical role of the J-domain in stimulating Hsp70 ATPase activity and the functional diversity of Hsp70. Furthermore, it explores the interactions between the G/F region of Sis1 and the J-domain, shedding light on the fine-tuning of the Hsp70-substrate cycle.