Background: Currently, no information is available about the association of the mitochondrial porin pore with the major outer membrane proteins Om14p and Om45p. Results: Por1p forms complexes with Om14p and Om45p. Conclusion: Molecular organization of the porin pore and its interaction with the inner membrane is influenced by Om14p and Om45p. Significance: The newly identified protein complex improves the understanding of mitochondrial transport processes.

In the yeast Saccharomyces cerevisiae, mitochondrial translation of most, if not all, mitochondrially encoded genes is regulated by an individual set of gene-specific activators. Translation of the COB mRNA encoding cytochrome b requires the function of two nuclearly encoded proteins, Cbs1p and Cbs2p. Genetic data revealed that the 5'-untranslated region of COB mRNA is the target of both proteins. Recently, we provided evidence for an interaction of Cbs2p with mitochondrial ribosomes. We demonstrate here by means of blue native gel electrophoresis, density gradient centrifugation and tandem affinity purification that a portion of Cbs1p is also associated with mitochondrial ribosomes. In addition, we demonstrate that the amount of ribosome-associated Cbs1p is elevated in the presence of chloramphenicol, which is known to stall ribosomes on mRNAs. In the presence of puromycin, which strips off the mRNA and nascent protein chains from ribosomes, Cbs1p is no longer associated with ribosomes. Our data indicate that the observed interaction is mediated by ribosome-bound mRNA, thus restricting the association to ribosomes actively translating cytochrome b.

This is a response to a letter by Rapaport (1).We thank Dr. Rapaport for his interest in our studies. Yaffe et al. (2) didn't present experimental data on the topology of the protein; instead they cite Riezman et al. (3) ("Previous studies…indicate that OM45 is an integral membrane protein with the bulk … exposed to the cytoplasm."). However, Riezman et al. (3) state: "We conclude … that this 45-kDa protein is exposed to the intermembrane space in intact mitochondrion…."Besides immunological data, the most convincing experiment shows a Coomassie-stained gel of separated OM-vesicle proteins with/without trypsin treatment (Fig. 7 in Ref. 3). Upon trypsin treatment the 70-kDa band (Tom70p) is completely removed, as expected. In contrast, intensity of the 45-kDa band, most likely representing Om45p, is not altered at all. This is a clear indication that this protein is not accessible for trypsin and protrudes into the inner membrane space (IMS). In addition, topology prediction programs strongly favor (4) or unambiguously assign (5) the "N terminus outside" orientation.We always "calibrate" the required proteinase K concentration by repeated experiments with serial enzyme dilutions. We presented only the results for a concentration of 2 g/ml. At 5 g/ml matrix control proteins start to be degraded without membrane disruption, whereas 0.5 g/ml showed almost no proteolytic effect. In addition, we always use Triton X-100 to document that the proteins are not protease-resistant per se. This important control is missing in Sesaki and Jensen (6).In conclusion, our data on the topology of cMyc-tagged Om45p are consistent with an exposure to the IMS, as proposed by Riezman et al. (3) for the untagged protein.