The structural basis by which Hsp104 dissolves disordered aggregates and prions 
is unknown. A single subunit within the Hsp104 hexamer can solubilize disordered 
aggregates, whereas prion dissolution requires collaboration by multiple Hsp104 
subunits. Here, we establish that the poorly understood Hsp104 N-terminal domain 
(NTD) enables this operational plasticity. Hsp104 lacking the NTD (Hsp104(ΔN)) 
dissolves disordered aggregates but cannot dissolve prions or be potentiated by 
activating mutations. We define how Hsp104(ΔN) invariably stimulates Sup35 
prionogenesis by fragmenting prions without solubilizing Sup35, whereas Hsp104 
couples Sup35 prion fragmentation and dissolution. Volumetric reconstruction of 
Hsp104 hexamers in ATPγS, ADP-AlFx (hydrolysis transition state mimic), and ADP 
via small-angle X-ray scattering revealed a peristaltic pumping motion upon ATP 
hydrolysis, which drives directional substrate translocation through the central 
Hsp104 channel and is profoundly altered in Hsp104(ΔN). We establish that the 
Hsp104 NTD enables cooperative substrate translocation, which is critical for 
prion dissolution and potentiated disaggregase activity.