Conformational Dynamics of the Mitochondrial TIM23 Preprotein Translocase

The vast majority of mitochondrial proteins are synthesized by the
cytosolic ribosomes as precursor proteins which have to be
transported into the organelle to reach their sites of function.
The whole process of recognition, translocation,
intra-mitochondrial sorting of and assembly of precursor proteins
is achieved by the concerted action of different mitochondrial
translocases. All proteins destined for the mitochondrial matrix
and some inner membrane proteins are imported first by the TOM
complex of the outer membrane and subsequently by the TIM23 complex
of the inner membrane in an energy-driven process. The TIM23
complex was found to consist of ten components, conventionally
divided into two sectors: membrane sector harbouring the
translocation channel and the import motor on the matrix side of
the membrane sector. In the first part of the present work, the two
most recently discovered subunits of the TIM23 complex, Pam17 and
Tim21 were characterized. A systematic characterization revealed
that both of these non-essential subunits of the translocase are
associated with Tim17-Tim23 core of the membrane sector of the
TIM23 translocase. A functional connection between the two
non-essential components was discovered. Results presented in this
part showed that Pam17 and Tim21 modulate the functions of the
TIM23 complex in an antagonistic manner. The second part of the
work was directed towards understanding the motor sector of the
translocase in terms of the regulated interaction between Tim44 and
Ssc1. Previous studies on the Tim44:Ssc1 interaction were able to
discern the steady-state properties of Tim44:Ssc1 interaction in
organello and in vitro. However, due to the limitations of the
techniques used, they were unable to shed light on the kinetics and
dynamics of the process. The translocation event is a dynamic event
with conformational cycling of the various components. Therefore,
the kinetic components essential in defining the cycle of events in
the motor sector were explored. A FRET based assay to analyze the
Tim44:Ssc1 interaction in real time was developed. The same set of
tools was also used to resolve the regions of the two proteins that
determine their interaction. The substrate induced dissociation of
Tim44:Ssc1 complex was found to be too slow to support a
physiological rate of protein translocation. ATP-induced
dissociation was observed to be fast enough to be physiologically
relevant. The dissociation of Ssc1 from Tim44 occurred in a one
step manner without Tim44 anchored conformational changes.
Furthermore, peptide-array scanning of mitochondrial matrix
proteins revealed that Ssc1 and Tim44 share complementary binding
sites on the precursor proteins which could prevent backsliding of
preproteins. The data support the Brownian ratchet model mediated
translocation of preproteins into the mitochondrial matrix. The
third part of the work aimed at dissecting the chaperone cycle of
Ssc1 in the mitochondrial matrix, in terms of conformational
changes and binding of co-chaperones. Using the FRET sensors
developed, the inter-domain conformation and lid-base conformations
of the PBD of Ssc1 could be investigated. Single particle FRET
(SpFRET) analysis showed that in the ATP-bound form Ssc1 populates
a homogeneous conformational state with respect to the inter-domain
conformation and conformation of the lid to base of the PBD. On the
contrary, in the ADP-bound state the conformation of the chaperone
is heterogenous. Using the same sensors on bacterial homologue
DnaK, specific differences in conformational distributions were
observed. Furthermore, the active role of substrates in determining
the inter-domain conformation and lid-closing was evident from the
SpFRET based conformational analyses. Using ensemble time resolved
FRET, the kinetics and dynamics of conformational changes along
with binding of co-chaperones were explored. This provided a better
understanding of the conformational dynamics of Ssc1 in the context
of functional chaperone cycle in the mitochondrial matrix.