Abstract
The co-chaperone Hep1 is required to prevent the aggregation of mitochondrial Hsp70 proteins. We have analyzed the interaction of Hep1 with mitochondrial Hsp70 (Ssc1) and the determinants in Ssc1 that make it prone to aggregation. The ATPase and peptide binding domain (PBD) of Hsp70 proteins are connected by a linker segment that mediates interdomain communication between the domains. We show here that the minimal Hep1 binding entity of Ssc1 consists of the ATPase domain and the interdomain linker. In the absence of Hep1, the ATPase domain with the interdomain linker had the tendency to aggregate, in contrast to the ATPase domain with the mutated linker segment or without linker, and in contrast to the PBD. The closest homolog of Ssc1, bacterial DnaK, and a Ssc1 chimera, in which a segment of the ATPase domain of Ssc1 was replaced by the corresponding segment from DnaK, did not aggregate in Delta hep1 mitochondria. The propensity to aggregate appears to be a specific property of the mitochondrial Hsp70 proteins. The ATPase domain in combination with the interdomain linker is crucial for aggregation of Ssc1. In conclusion, our results suggest that interdomain communication makes Ssc1 prone to aggregation. Hep1 counteracts aggregation by binding to this aggregation-prone conformer.
Highlights
To understand the mechanism of function of Hep1 we have studied its interaction with mitochondrial Hsp70 and with the various parts of mtHsp70, the ATPase domain, the peptide binding domain (PBD), and the interdomain linker
All Mitochondrial Hsp70 Family Members Have the Propensity to Aggregate—Mitochondrial Hsp70 proteins Ssc1 and Ssq1 have the tendency to aggregate in the absence of their co-chaperone Hep1 [28, 29]
To address whether all mitochondrial Hsp70 proteins are prone to aggregation we studied the third member of the Hsp70 family of yeast mitochondria, Ssc3/ Ecm10
Summary
To understand the mechanism of function of Hep1 we have studied its interaction with mitochondrial Hsp70 and with the various parts of mtHsp70, the ATPase domain, the PBD, and the interdomain linker. We show here that it is the ATPase domain together with the interdomain linker of Ssc1 that binds to Hep1 in mitochondria. The ATPase domain, the PBD with the interdomain linker at its N terminus, referred to as PBD, were expressed as His-tagged variants in wild type cells and tested for interaction with Hep1.
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