Expression, purification, and in vitro characterization of the human outer mitochondrial membrane receptor human translocase of the outer mitochondrial membrane 20.
In order to investigate the biochemical properties of the mitochondrial outer membrane receptor, hTom20, involved in protein recognition, the cytosolic domain of this receptor was overexpressed and purified to homogeneity. A four-step purification including the purification of thrombin is described as well as an analysis of the function of the highly purified hTom20 protein. The receptor was concentrated and the subsequent aggregation behavior was investigated in order to understand the function of the single cysteine in the cytosolic domain as well as the function of the proposed "glutamine face" for the structure of the protein. It was found that specific dimerization of the cytosolic domain of hTom20 is necessary in order to prevent aggregation of the protein. In addition, the cysteine and the glutamine face are important for the stability of the protein. We propose that the function of the cysteine is to promote dimerization as found in the absence of dithiothreitol.
Schleiff
E
E
Department of Biochemistry, McGill University, 3655 Drummond, Montreal, H3G 1Y6, Canada. eschleiff@bot.uni-kiel.de
Khanna
R
R
Orlicky
S
S
Vrielink
A
A
D019943:Amino Acid Substitution; D001665:Binding Sites; D003545:Cysteine / Q000235:genetics / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism; D003902:Detergents / Q000494:pharmacology; D019281:Dimerization; D005973:Glutamine / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007425:Intracellular Membranes / Q000378:metabolism*; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D026901:Membrane Transport Proteins; D008928:Mitochondria / Q000378:metabolism*; D000090622:Mitochondrial Precursor Protein Import Complex Proteins; D010446:Peptide Fragments / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D011487:Protein Conformation / Q000187:drug effects; D017510:Protein Folding; D011956:Receptors, Cell Surface; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D019163:Reducing Agents / Q000494:pharmacology; D017384:Sequence Deletion; D013816:Thermodynamics; D013917:Thrombin / Q000378:metabolism; D014270:Trifluoroethanol / Q000494:pharmacology
D003902:Detergents; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D000090622:Mitochondrial Precursor Protein Import Complex Proteins; D010446:Peptide Fragments; D011956:Receptors, Cell Surface; D011993:Recombinant Fusion Proteins; D019163:Reducing Agents; C084008:TOMM20 protein, human; D005973:Glutamine; D014270:Trifluoroethanol; D013917:Thrombin; D003545:Cysteine