Molecular chaperones of the Hsp70 class are essential for a number
of cellular processes. The yeast mitochondrial Hsp70 chaperone Ssc1
plays an indispensable role for the mitochondrial biogenesis. As an
essential component of the import motor of the TIM23 transolcase,
Ssc1 drives the ATP-dependent translocation of proteins into the
mitochondrial matrix. Moreover, it mediates the de novo folding and
the assembly of several proteins in the mitochondrial matrix and
prevents the formation of protein aggregates. Surprisingly, Ssc1
itself has a propensity to self-aggregate. Thus, it requires a
helper protein, the chaperone Hep1 that prevents Ssc1 aggregation
and maintains its structure and function. The mechanism of the
protective function of Hep1 on Ssc1, however, is not understood. In
the present study, the structural determinants of Ssc1 that make it
prone to aggregation and the structural requirements of Ssc1 for
its interaction with Hep1 were analysed and provided insights into
the mechanism of prevention of Ssc1 aggregation by Hep1. The
aggregation studies demonstrate that a variant of Ssc1 consisting
of the ATPase domain and the subsequent interdomain linker
aggregates in absence of Hep1. In contrast, the PBD and the ATPase
domain alone are not prone to aggregation. Moreover, the
interaction studies reveal that the aggregation-prone region seems
to be the smallest entity within Ssc1 required for the interaction
with Hep1. Taken together, the native Ssc1 adopts an
aggregation-prone conformation, in which the ATPase domain with the
interdomain linker has the propensity to aggregate. Hep1 binds to
this aggregation-prone region and thereby counteracts the
aggregation process and keeps the native Ssc1 in a functional and
active state. Although Hsp70 chaperones are important for the
biogenesis of a multitude of proteins, little is known about the
biogenesis of these chaperones themselves. The present study
reports on the analysis of the folding process of the mitochondrial
Hsp70 chaperone Ssc1. In organello, in vivo and in vitro assays
were established and then employed to study the de novo folding of
Ssc1. Upon import into mitochondria, Ssc1 folds rapidly with the
ATPase domain and the PBD adopting their structures independently
of each other. Notably, the ATPase domain requires the presence of
the interdomain linker for its folding, whereas the PBD folds
without the linker. Moreover, in the absence of Hep1, the ATPase
domain with the interdomain linker displays a severe folding
defect, which indicates a role of Hep1 in the folding process of
Ssc1. Apart from Hep1, none of the general mitochondrial chaperone
systems seem to be important for the folding of Ssc1. Furthermore,
the folding process of Ssc1 was reconstituted in vitro and the main
steps of the folding pathway of Ssc1 were characterised. Hep1 and
ATP/ADP are required and sufficient for the folding of Ssc1 into
the native, catalytically active form. In an early step of folding,
Hep1 interacts with the folding intermediate of Ssc1. This
interaction induces conformational changes which allow binding of
ATP/ADP. The binding of a nucleotide triggers Hep1 release and
further folding of the intermediate into a native Ssc1. The present
study provides the first direct evidence for the requirement of
Hep1 for the folding of the Ssc1 chaperone. Thus, it demonstrates
for the first time that the de novo folding of an Hsp70 chaperone
depends on a specialized proteinaceous factor. In conclusion, Hep1
fulfils a dual chaperone function in the cell. It mediates the de
novo folding of Ssc1 and maintains folded Ssc1 in a functional
state during the ATPase cycle. Therefore, the Hep1 chaperone plays
a crucial role for the protein biogenesis and homeostasis in
mitochondria.
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