The Mitochondrial Protein OM45 Is Exposed to the Cytosol

This letter concerns the article published recently by Lauffer et al. (1). OM45 is an abundant protein of the outer membrane (OM) of yeast mitochondria. Yaffe et al. (2) suggested that the protein is anchored to the OM via a single transmembrane segment located at the N terminus of the protein whereas the bulk of the protein is exposed to the cytosol. Later reports confirmed that the protein is exposed to externally added trypsin (3). Furthermore, we observed that OM45 shares its biogenesis pathway with other proteins with such a topology (4). In contrast, Lauffer et al. (1) reported that OM45 has an opposite orientation where the bulk of the protein faces the mitochondrial intermembrane space. The authors based their claim on the resistance of the protein to a very low concentration of externally added proteinase K. However, it is well known that resistance to one protease (especially with the low concentration used by Lauffer et al. (1)) does not exclude availability to other proteases. Thus, their conclusion is based on possibly inadequate experimental assay, and they are advised to repeat their assay with increasing amounts of both trypsin and proteinase K. Of note, Lauffer et al. (1) cited a report by Riezman et al. (5) as supporting their claim. However, Riezman et al. (5) used nonspecific antibodies against proteins of 45 kDa, and only one of the two identified proteins was resistant to trypsin. Taken together, the correct orientation of OM45 is such that the bulk of the protein is facing the cytosol, and the arguments presented by Lauffer et al. (1) are not convincing enough to claim otherwise.