Reply to Rapaport: The Mitochondrial Protein OM45 Is Exposed to the Cytosol

Abstract

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.

A Cross-linking Mass Spectrometry Approach Defines Protein Interactions in Yeast Mitochondria

“…Om45 has originally been described as an outer membrane protein, facing into the cytosol ( 87 , 88 ), although IMS localization has also been reported ( 89 ). We found that Om45 extensively cross-links to porin, which is in accordance with a previous biochemical study ( 89 , 110 ). We also show that Om45 extensively cross-links to Nde1 suggesting that it forms a complex.…”

Records of RNA locations in living yeast revealed through covalent marks

“…To detect RNAs near the mitochondrial outer membrane, we inserted PUP-2 downstream of the OM45 gene, resulting in an Om45p–PUP-2 chimera, which we refer to as “Mito-PUP” hereafter. Om45p is predicted to be cotranslationally inserted into the mitochondrial membrane with its C-terminal PUP-2 domain in the cytosol ( 37 – 42 ) ( Fig. 3 A ).…”

“…( A ) Architecture of Mito-PUP. PUP alone was fused to the cytosolic C-terminal end of the mitochondrial membrane protein, Om45p ( 37 – 42 ). ( B ) Confocal localization of Mito-PUP fluorescence (green) and the mitochondrial marker, Tom70p-mCherry ( 31 ) (red), including fluorescence intensity across a line that bisects the cell (below).…”

Role of the small protein Mco6 in the mitochondrial sorting and assembly machinery

“…40,46–48 After identification of the a-helical membrane proteins of the TOM complex, the specific complex formation between β-barrel membrane proteins and a-helical membrane proteins was regarded as a special case. Meanwhile the porin β-barrel membrane protein was found to interact with the a-helical membrane proteins Om14 and Om45 and the β-barrel protein Mdm10 interacts as subunit of the ER-mitochondria encounter structure (ERMES) with the a-helical outer membrane proteins Tom7 and Gem1(Miro-1) 23,25–27,49…”