Normal cellular functions rely on correct protein localization within cells. Protein targeting had been thought to be a precise process, and even if it fails, the mistargeted proteins were supposed ...to be quickly degraded. However, this view is rapidly changing. Tail-anchored (TA) proteins are a class of membrane proteins that possess a single transmembrane domain (TMD) near the C-terminus and are posttranslationally targeted to the endoplasmic reticulum (ER) membrane, mitochondrial outer membrane (OM), and peroxisomal membrane, yet they can be mistargeted to the mitochondrial OM. The mistargeted TA proteins can be extracted from the OM by a mitochondrial AAA-ATPase Msp1/ATAD1 and transferred to the ER. If they are regarded as aberrant by the ER protein quality control system, they are extracted from the ER membrane for proteasomal degradation in the cytosol. If they are not regarded as aberrant, they are further transported to downstream organelles or original destinations along the secretory pathway. Thus, Msp1 contributes to not only degradation but also "proofreading" of the targeting of mislocalized TA proteins.
Abstract
Since the 1950s, electron microscopic observations have suggested the existence of special regions where the distinct organelle membranes are closely apposed to each other, yet their ...molecular basis and functions have not been examined for a long time. Recent studies using yeast as a model organism identified multiple organelle-membrane tethering sites/factors, such as ERMES (ER–mitochondria encounter structure), NVJ (Nuclear–vacuole junction), vCLAMP (Vacuole and mitochondria patch) and MICOS (Mitochondrial contact site). Among them, ERMES is the best-characterized contact-site protein complex, which was found to function as not only an organelle-tethering factor but also a phospholipid transfer protein complex. In this review, we will discuss recent advances in the characterization of ERMES and other organelle contact zones, vCLAMP, NVJ and MICOS in yeast.
Mitochondria are surrounded by the two membranes, the outer and inner membranes, whose lipid compositions are optimized for proper functions and structural organizations of mitochondria. Although a ...part of mitochondrial lipids including their characteristic lipids, phosphatidylethanolamine and cardiolipin, are synthesized within mitochondria, their precursor lipids and other lipids are transported from other organelles, mainly the ER. Mitochondrially synthesized lipids are re-distributed within mitochondria and to other organelles, as well. Recent studies pointed to the important roles of inter-organelle contact sites in lipid trafficking between different organelle membranes. Identification of Ups/PRELI proteins as lipid transfer proteins shuttling between the mitochondrial outer and inner membranes established a part of the molecular and structural basis of the still elusive intra-mitochondrial lipid trafficking.
Mitochondria are essential eukaryotic organelles responsible for primary cellular energy production. Biogenesis, maintenance, and functions of mitochondria require correct assembly of resident ...proteins and lipids, which require their transport into and within mitochondria. Mitochondrial normal functions also require an exchange of small metabolites between the cytosol and mitochondria, which is primarily mediated by a metabolite channel of the outer membrane (OM) called porin or voltage-dependent anion channel. Here, we describe recently revealed novel roles of porin in the mitochondrial protein and lipid transport. First, porin regulates the formation of the mitochondrial protein import gate in the OM, the translocase of the outer membrane (TOM) complex, and its dynamic exchange between the major form of a trimer and the minor form of a dimer. The TOM complex dimer lacks a core subunit Tom22 and mediates the import of a subset of mitochondrial proteins while the TOM complex trimer facilitates the import of most other mitochondrial proteins. Second, porin interacts with both a translocating inner membrane (IM) protein like a carrier protein accumulated at the small TIM chaperones in the intermembrane space and the TIM22 complex, a downstream translocator in the IM for the carrier protein import. Porin thereby facilitates the efficient transfer of carrier proteins to the IM during their import. Third, porin facilitates the transfer of lipids between the OM and IM and promotes a back-up pathway for the cardiolipin synthesis in mitochondria. Thus, porin has roles more than the metabolite transport in the protein and lipid transport into and within mitochondria, which is likely conserved from yeast to human.
Newly synthesized mitochondrial precursor proteins have to become unfolded to cross the mitochondrial membranes. This unfolding is achieved primarily by mitochondrial Hsp70 (mtHsp70) for ...presequence-containing precursor proteins. However, the membrane potential across the inner membrane (ΔΨ) could also contribute to unfolding of short-presequence containing mitochondrial precursor proteins. Here we investigated the role of ΔΨ in mitochondrial protein unfolding and import. We found that the effects of mutations in the presequence on import rates are correlated well with the hydrophobicity or ability to interact with import motor components including mtHsp70, but not with ΔΨ (negative inside). A spontaneously unfolded precursor protein with a short presequence is therefore trapped by motor components including mtHsp70, but not ΔΨ, which could cause global unfolding of the precursor protein. Instead, ΔΨ may contribute the precursor unfolding by holding the presequence at the inner membrane for trapping of the unfolded species by the import motor system.
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.
The cryoelectron microscopy structures of the mitochondrial protein import gate, the translocase of the outer mitochondrial membrane (TOM) complex, have become available. The yeast TOM complex structure shows the presence of two paths within the import channel for different classes of mitochondrial precursor proteins. This explains how the TOM import channel can handle and translocate ˜ 1000 different precursor proteins with different physical and chemical properties from the cytosol to the mitochondrial interior across the outer membrane.
Mitochondria are two-membrane bounded organelles consisting of 1000–2000 different proteins, most of which are synthesized in the cytosol and subsequently imported into mitochondria. The imported ...proteins are further sorted to one of the four compartments, the outer membrane, intermembrane space, inner membrane, and matrix, mostly following one of the five major pathways. Mitochondrial protein import and sorting are mediated by the translocator complexes in the membranes and chaperones in the aqueous compartments operating along the import pathways. Here, we summarize the expanding knowledge on the roles of translocators, chaperones, and related components in the multiple pathways for mitochondrial protein trafficking.
As phospholipids are synthesized mainly in the endoplasmic reticulum (ER) and mitochondrial inner membranes, how cells properly distribute specific phospholipids to diverse cellular membranes is a ...crucial problem for maintenance of organelle-specific phospholipid compositions. Although the ER-mitochondria encounter structure (ERMES) was proposed to facilitate phospholipid transfer between the ER and mitochondria, such a role of ERMES is still controversial and awaits experimental demonstration. Here we developed a novel in vitro assay system with isolated yeast membrane fractions to monitor phospholipid exchange between the ER and mitochondria. With this system, we found that phospholipid transport between the ER and mitochondria relies on membrane intactness, but not energy sources such as ATP, GTP or the membrane potential across the mitochondrial inner membrane. We further found that lack of the ERMES component impairs the phosphatidylserine transport from the ER to mitochondria, but not the phosphatidylethanolamine transport from mitochondria to the ER. This in vitro assay system thus offers a powerful tool to analyze the non-vesicular phospholipid transport between the ER and mitochondria.
Most mitochondrial proteins are synthesized as precursor proteins (preproteins) in the cytosol and imported into mitochondria. The translocator of the outer membrane (TOM) complex functions as a main ...entry gate for the import of mitochondrial proteins. The TOM complex is a multi-subunit membrane protein complex composed of a β-barrel channel Tom40 and six single-pass membrane proteins. Recent cryo-EM studies have revealed high-resolution structures of the yeast and human TOM complexes, which enabled us to discuss the mechanism of protein import at an amino-acid residue level. The cryo-EM structures show that two Tom40 β-barrels are surrounded by two sets of small Tom subunits to form a dimeric structure. The intermembrane space (IMS) domains of Tom40, Tom22, and Tom7 form a binding site for presequence-containing preproteins in the middle of the dimer to achieve their efficient transfer of to the downstream translocase, the TIM23 complex. The N-terminal segment of Tom40 spans the channel from the cytosol to the IMS to interact with Tom5 at the periphery of the dimer, where downstream components of presequence-lacking preproteins are recruited. Structure-based biochemical analyses together with crosslinking experiments revealed that each Tom40 channel possesses two distinct paths and exit sites for protein translocation of different sets of mitochondrial preproteins. Here we summarize the current knowledge on the structural features, protein translocation mechanisms, and remaining questions for the TOM complexes, with particular emphasis on their determined cryo-EM structures. This article is an extended version of the Japanese article, Structural basis for protein translocation by the translocase of the outer mitochondrial membrane, published in SEIBUTSU BUTSURI Vol. 60, p. 280-283 (2020).
Functional integrity of eukaryotic organelles relies on direct physical contacts between distinct organelles. However, the entity of organelle-tethering factors is not well understood due to lack of ...means to analyze inter-organelle interactions in living cells. Here we evaluate the split-GFP system for visualizing organelle contact sites in vivo and show its advantages and disadvantages. We observed punctate GFP signals from the split-GFP fragments targeted to any pairs of organelles among the ER, mitochondria, peroxisomes, vacuole and lipid droplets in yeast cells, which suggests that these organelles form contact sites with multiple organelles simultaneously although it is difficult to rule out the possibilities that these organelle contacts sites are artificially formed by the irreversible associations of the split-GFP probes. Importantly, split-GFP signals in the overlapped regions of the ER and mitochondria were mainly co-localized with ERMES, an authentic ER-mitochondria tethering structure, suggesting that split-GFP assembly depends on the preexisting inter-organelle contact sites. We also confirmed that the split-GFP system can be applied to detection of the ER-mitochondria contact sites in HeLa cells. We thus propose that the split-GFP system is a potential tool to observe and analyze inter-organelle contact sites in living yeast and mammalian cells.
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