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.
Beddoe
Travis
T
Protein Crystallography Unit, The ARC Centre for Structural and Functional Microbial Genomics, School of Biomedical Sciences, Monash University, Clayton, Victoria 3800, Australia.
Bushell
Simon R
SR
Perugini
Matthew A
MA
Lithgow
Trevor
T
Mulhern
Terrence D
TD
Bottomley
Stephen P
SP
Rossjohn
Jamie
J
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