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Recently, we found that myoglobin (Mb) localizes in both the cytosol and mitochondrial intermembrane space in rodent skeletal muscle. Most proteins of the intermembrane space pass through the outer mitochondrial membrane via the translocase of the outer membrane (TOM) complex. However, whether the TOM complex imports Mb remains unknown. The purpose of this study was to investigate the involvement of the TOM complex in Mb import into the mitochondria. A proteinase K protection assay of mitochondria from C2C12 myotubes confirmed that Mb integrated into the mitochondria. An immunoprecipitation assay verified the interaction of Mb and TOM complex receptors (Tom20, Tom70) in isolated mitochondria. The assay showed a clear interaction of Mb with Tom20 and Tom70. A knockdown experiment using siRNA for TOM complex receptors (Tom20, Tom70) and TOM complex channel (Tom40) did not alter the amount of Mb expression in the mitochondrial fraction. These results suggested that Mb does not necessarily require the TOM complex for mitochondrial import of Mb. Although the physiological role of Mb interactions with TOM complex receptors remains unclear, further studies are needed to clarify how Mb enters the mitochondria independently of the TOM complex.

The translocase of the outer mitochondrial membrane (TOM) is the main entry gate for proteins.sup.1-4. Here we use cryo-electron microscopy to report the structure of the yeast TOM core complex.sup.5-9 at 3.8-Å resolution. The structure reveals the high-resolution architecture of the translocator consisting of two Tom40 [beta]-barrel channels and [alpha]-helical transmembrane subunits, providing insight into critical features that are conserved in all eukaryotes.sup.1-3. Each Tom40 [beta]-barrel is surrounded by small TOM subunits, and tethered by two Tom22 subunits and one phospholipid. The N-terminal extension of Tom40 forms a helix inside the channel; mutational analysis reveals its dual role in early and late steps in the biogenesis of intermembrane-space proteins in cooperation with Tom5. Each Tom40 channel possesses two precursor exit sites. Tom22, Tom40 and Tom7 guide presequence-containing preproteins to the exit in the middle of the dimer, whereas Tom5 and the Tom40 N extension guide preproteins lacking a presequence to the exit at the periphery of the dimer. The high-resolution cryo-electron microscopy structure of the yeast translocase of the outer mitochondrial membrane reveals key features of mitochondrial protein import that are conserved in all eukaryotes., Author(s): Yuhei Araiso [sup.1] [sup.2] [sup.12] , Akihisa Tsutsumi [sup.3] , Jian Qiu [sup.4] [sup.5] [sup.13] , Kenichiro Imai [sup.6] , Takuya Shiota [sup.7] [sup.8] , Jiyao Song [sup.4] , [...]

Pyrrolysine (pyl), the 22nd natural amino acid, is genetically encoded by UAG and inserted into proteins by the unique suppressor [tRNA.sup.Pyl] (ref. 1). The Methanosarcinaceae produce Pyl and express Pyl-containing methyltransferases that allow growth on methylamines (2). Homologous methyltransferases and the Pyl biosynthetic and coding machinery are also found in two bacterial species (1,3). Pyl coding is maintained by pyrrolysyl-tRNA synthetase (PylRS), which catalyses the formation of Pyl-[tRNA.sup.Pyl] (refs 4, 5). Pyl is not a recent addition to the genetic code. PylRS was already present in the last universal common ancestorb; it then persisted in organisms that utilize methylamines as energy sources. Recent protein engineering efforts added non-canonical amino acids to the genetic code (7,8). This technology relies on the directed evolution of an 'orthogonal' tRNA synthetase-tRNA pair in which an engineered aminoaryl-tRNA synthetase (aaRS) specifically and exclusively arylates the orthogonal tRNA with a non-canonical amino acid. For Pyl the natural evolutionary process developed such a system some 3 billion years ago. When transformed into Escherichia coli, Methanosarcina barkeri PylRS and [tRNA.sup.Pyl] function as an orthogonal pair in vivos (5,9). Here we show that Desulfitobacterium hafniense PylRS-[tRNA.sup.Pyl] is an orthogonal pair in vitro and in vivo, and present the crystal structure of this orthogonal pair. The ancient emergence of PylRS-[tRNA.sup.Pyl] allowed the evolution of unique structural features in both the protein and the tRNA. These structural elements manifest an intricate, specialized aaRS-tRNA interaction surface that is highly distinct from those observed in any other known aaRS-tRNA complex; it is this general property that underlies the molecular basis of orthogonality., Unlike the archaeal PylRS sequences, the bacterial versions are encoded in two separate genes. The pylS gene encodes the D. hafniense PylRS (DhPylRS) presented here, which includes a tRNA recognition [...]

The translocase of the outer mitochondrial membrane (TOM) complex is the main entry gate for most mitochondrial proteins. The TOM complex is a multisubunit membrane protein complex consisting of a β‐barrel protein Tom40 and six α‐helical transmembrane (TM) proteins, receptor subunits Tom20, Tom22, and Tom70, and regulatory subunits Tom5, Tom6, and Tom7. Although nearly 30 years have passed since the main components of the TOM complex were identified and characterized, the structural details of the TOM complex remained poorly understood until recently. Thanks to the rapid development of the cryoelectron microscopy (EM) technology, high‐resolution structures of the yeast TOM complex have become available. The identified structures showed a symmetric dimer containing five different subunits including Tom22. Biochemical and mutational analyses based on the TOM complex structure revealed the presence of different translocation paths within the Tom40 import channel for different classes of translocating precursor proteins. Previous studies including our cross‐linking analyses indicated that the TOM complex in intact mitochondria is present as a mixture of the trimeric complex containing Tom22. Furthermore, the dimeric complex lacking Tom22, and the trimer and dimer may handle different sets of mitochondrial precursor proteins for translocation across the outer membrane. In this Structural Snapshot, we will discuss possible rearrangement of the subunit interactions upon dynamic conversion of the TOM complex between the different subunit assembly states, the Tom22‐containing core dimer and trimer. [ABSTRACT FROM AUTHOR]

Pyrrolysine (Pyl), the 22nd natural amino acid, is genetically encoded by UAG and inserted into proteins by the unique suppressor tRNAPyl (ref. 1). The Methanosarcinaceae produce Pyl and express Pyl-containing methyltransferases that allow growth on methylamines. Homologous methyltransferases and the Pyl biosynthetic and coding machinery are also found in two bacterial species. Pyl coding is maintained by pyrrolysyl-tRNA synthetase (PylRS), which catalyses the formation of Pyl-tRNAPyl (refs 4, 5). Pyl is not a recent addition to the genetic code. PylRS was already present in the last universal common ancestor; it then persisted in organisms that utilize methylamines as energy sources. Recent protein engineering efforts added non-canonical amino acids to the genetic code. This technology relies on the directed evolution of an ‘orthogonal’ tRNA synthetase–tRNA pair in which an engineered aminoacyl-tRNA synthetase (aaRS) specifically and exclusively acylates the orthogonal tRNA with a non-canonical amino acid. For Pyl the natural evolutionary process developed such a system some 3 billion years ago. When transformed into Escherichia coli, Methanosarcina barkeri PylRS and tRNAPyl function as an orthogonal pair in vivo. Here we show that Desulfitobacterium hafniense PylRS–tRNAPyl is an orthogonal pair in vitro and in vivo, and present the crystal structure of this orthogonal pair. The ancient emergence of PylRS–tRNAPyl allowed the evolution of unique structural features in both the protein and the tRNA. These structural elements manifest an intricate, specialized aaRS–tRNA interaction surface that is highly distinct from those observed in any other known aaRS–tRNA complex; it is this general property that underlies the molecular basis of orthogonality. [ABSTRACT FROM AUTHOR]

Recent developments in cryo-electron microscopy techniques have facilitated intensive research into determining protein structures. Nevertheless, the structures of some mitochondrial membrane protein complexes remain undetermined. One possible reason for this research gap is that mitochondrial membrane protein complexes are difficult to overexpress and purify. Even using high-resolution cryo-electron microscopy, structural determination is not possible without first obtaining purified homogeneous proteins. As determining novel structures of protein complexes would provide opportunities to answer many unresolved biological questions, it is important to generalize purification methods, which often become bottlenecks in protein research. In this chapter, we introduce purification methods for mitochondrial membrane protein complexes and mitochondria-localized soluble protein complexes using a yeast expression system. We also describe the recent development of a mitochondrial membrane isolation method that enables the extraction of large amounts of protein complexes for structural analyses., (Copyright © 2024. Published by Elsevier Inc.)

Objective: Protein S(PS) activity, especially PS-specific activity calculated by total PS activity (tPSAct) divided by total PS antigen (tPSAg), is important in the diagnosis of hereditary PS deficiency (PSD). The cleavage of PS at a thrombin-sensitive region (TSR) by proteases reduces the anticoagulant activity of PS. Therefore, we investigated the effect of sample processing and storage on tPSAct and PS cleavage., Methods: Blood samples were collected from ten healthy subjects, and tPSAg and tPSAct were measured in whole blood or plasma stored at room temperature (RT) or 4°C. The cleaved PS was detected by western blotting, and the relationship between decreases in PS-specific activity and increase rates of cleaved PS was evaluated. Furthermore, the stability of tPSAg and tPSAct on the long-term storage of plasma was also evaluated., Results: Both whole blood and plasma stored at RT and whole blood stored at 4°C showed decreased tPSAct (50-80%) after 24 hours ( P <0.05). PS-specific activity levels negatively correlated with the increase rate of cleaved PS (r =-0.84, P <0.001). The tPSAct was decreased to 60% after three days in plasma stored at 4°C ( P <0.05) but was stable for about one month when stored at -20°C or below., Conclusion: Inappropriate processing and storage result in falsely low PS-specific activity due to the cleavage of PS in the blood collection tubes, which may lead to misdiagnosis of PSD. Samples should be centrifuged immediately after collection, and the plasma should be frozen., (© 2021 by the Association of Clinical Scientists, Inc.)