A biophysical analysis of the tetratricopeptide repeat-rich mitochondrial import receptor, Tom70, reveals an elongated monomer that is inherently flexible, unstable, and unfolds via a multistate pathway.
Proteins destined for all submitochondrial compartments are translocated across the outer mitochondrial membrane by the TOM (translocase of the outer membrane) complex, which consists of a number of specialized receptor subunits that bind mitochondrial precursor proteins for delivery into the translocation channel. One receptor, Tom70, binds large, hydrophobic mitochondrial precursors. The current model of Tom70-mediated import involves multiple dimers of the receptor recognizing a single molecule of substrate. Here we show via a battery of biophysical and spectroscopic techniques that the cytosolic domain of Tom70 is an elongated monomer. Thermal and urea-induced denaturation revealed that the receptor, which unfolds via a multistate pathway, is a relatively unstable molecule undergoing major conformational change at physiological temperatures. The data suggest that the malleability of the monomeric Tom70 receptor is an important factor in mitochondrial import.
Protein Crystallography Unit, The ARC Centre for Structural and Functional Microbial Genomics, School of Biomedical Sciences, Monash University, Clayton, Victoria 3800, Australia.
Travis
T
Beddoe
Simon R
SR
Bushell
Matthew A
MA
Perugini
Trevor
T
Lithgow
Terrence D
TD
Mulhern
Stephen P
SP
Bottomley
Jamie
J
Rossjohn
D020816:Amino Acid Motifs; D001665:Binding Sites; D002942:Circular Dichroism; D003600:Cytosol / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D006358:Hot Temperature; D008024:Ligands; D008565:Membrane Proteins / Q000502:physiology*; D008928:Mitochondria / Q000378:metabolism*; D033681:Mitochondrial Membrane Transport Proteins; D000090622:Mitochondrial Precursor Protein Import Complex Proteins; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000502:physiology*; D013050:Spectrometry, Fluorescence; D013696:Temperature; D013816:Thermodynamics; D014461:Ultracentrifugation; D014508:Urea / Q000494:pharmacology
D008024:Ligands; D008565:Membrane Proteins; D033681:Mitochondrial Membrane Transport Proteins; D000090622:Mitochondrial Precursor Protein Import Complex Proteins; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; C489039:TOM70 protein, S cerevisiae; D014508:Urea; D004247:DNA