CRYSTAL 24 Abstract Submission Form

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A STRUCTURAL AND FUNCTIONAL CHARACTERISATION OF TOM70: A RECEPTOR OF
THE MITOCHONDRIAL IMPORT MACHINERY
Simon R. Bushell 1,2 , Travis Beddoe 1,2 , Matthew. A. Perugini 3 , Trevor Lithgow 3 ,
Terence D. Mulhern 3 , Stephen. P. Bottomley 2 , Jamie Rossjohn 1,2
1
Protein Crystallography Unit, The ARC Centre for Structural and Functional
Genomics; 2Dept. of Biochemistry and Molecular Biology, Monash University; 3Russell
Grimwade School of Biochemistry and Molecular Biology, University of Melbourne,
Parkville, Australia
Some 99% of mitochondria’s protein complement is encoded by the eukaryotic
organism’s nuclear genome and is imported into the organelle post-translationally with
the co-operation of various cytosolic chaperones such as MSF or Hsp70. The TOM
Complex (Translocase of the Outer Membrane) is a multi-subunit complex residing on
mitochondrial outer membrane. The translocase consists of several receptors (Tom70,
Tom20 and Tom22); the pore forming Tom40, and several other smaller subunits.
Proteins destined for the mitochondron’s inner membrane, such as carrier family
proteins, are usually targeted to the organelle via multiple internal targeting sequences
present in the precursor’s primary structure.
The receptor Tom70 recognises this class of chaperone-bound precursor proteins and
facilitates its translocation through the TOM complex’s import pore into the
intermembrane space, where its further localisation is facilitated by the TIM22 complex.
To better understand Tom70-mediated carrier import we recently performed a
biophysical characterisation of the receptor: the first such study for a component of the
mitochondrial import machinery. We demonstrated, via analytical ultra-centrifugation,
that Tom70 exists as a highly asymmetric monomer, incapable of dimerising
autonomously. Unfolding studies, as measured by circular dichroism and native
tryptophan fluorescence, show that the protein unfolds via a multi-step pathway and
undergoes major conformational change at physiological temperatures. This suggests
that Tom70 has discrete, differentially folding domains. Like other TPR-containing
proteins, Tom70’s inherent fluidity might suggest a coupled binding/folding mechanism
for client protein recognition.
Through further experiments, such as stopped-flow kinetics, tryptophan scanning, and
our on-going crystallographic analyses, we hope to gain further insight into the
organisation of the Tom70’s multi-domain structure and how its binding mechanism
actuates its role in the import of carrier proteins.
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