An outer envelope membrane component of the plastid protein import apparatus plays an essential role in Arabidopsis

T ranslocon at the o uter envelope membrane of c hloroplasts, 34  kDa (Toc34) is a GTP-binding component of the protein import apparatus within the outer envelope membrane of plastids. The Arabidopsis genome encodes two homologues of Toc34, designated atToc33 and atToc34. In this report, we describe the identification and characterization of two atToc34 knockout mutants, plastid protein import 3-1 ( ppi3-1 ) and ppi3-2 . Aerial tissues of the ppi3 mutants appeared similar to the wild type throughout development, and contained structurally normal chloroplasts that were able to efficiently import the Rubisco small subunit precursor (prSS) in vitro . The absence of an obvious ppi3 phenotype in green tissues presumably reflects the ability of atToc33 to substitute for atToc34 in the mutant, and the relatively high level of expression of the atTOC33 gene in these tissues. In the roots, where atTOC33 is expressed at a much lower level, significant growth defects were observed in both mutants: ppi3 roots were approximately 20–30% shorter than wild-type roots. Attempts to identify a double homozygote lacking atToc34 and atToc33 (by crossing the ppi3 mutants with ppi1 , an atToc33 knockout mutant) were unsuccessful, indicating that the function provided by atToc33/atToc34 is essential during early development. Plants that were homozygous for ppi1 and heterozygous for ppi3 displayed a chlorotic phenotype much more severe than that of the ppi1 single mutant. Furthermore, the siliques of these plants contained approximately 25% aborted seeds, indicating that the double homozygous mutation is embryo lethal. The data demonstrate that atToc33/atToc34 performs a central and essential role during plastid protein import, and indicate that the atToc34 isoform is relatively more important for plastid biogenesis in roots.     

Mechanisms of protein import across the mitochondrial outer membrane

Mitochondria import the majority of their proteins from the cytosol. At the mitochondrial outer membrane, import is initiated through a series of reactions, which include preprotein recognition, unfolding, insertion and translocation. These processes are facilitated by a multisubunit complex, the TOM complex. Specific roles can now be assigned to several components of this complex. Although the import machinery of the outer membrane can insert and translocate a few proteins on its own, completion of translocation o f most preproteins is dependent upon coupling to both the membrane potential and mt-Hsp70/ATP-driven transport across the inner membrane, mediated by the TIM complex.     

Functions of outer membrane receptors in mitochondrial protein import

Most mitochondrial proteins are synthesized in the cytosol as precursor proteins and are imported into mitochondria. The targeting signals for mitochondria are encoded in the presequences or in the mature parts of the precursor proteins, and are decoded by the receptor sites in the translocator complex in the mitochondrial outer membrane. The recently determined NMR structure of the general import receptor Tom20 in a complex with a presequence peptide reveals that, although the amphiphilicity and positive charges of the presequence is essential for the import ability of the presequence, Tom20 recognizes only the amphiphilicity, but not the positive charges. This leads to a new model that different features associated with the mitochondrial targeting sequence of the precursor protein can be recognized by the mitochondrial protein import system in different steps during the import.     

MAS6 encodes an essential inner membrane component of the yeast mitochondrial protein import pathway

To identify new components that mediate mitochondrial protein import, we analyzed mas6, an import mutant in the yeast Saccharomyces cerevisiae. mas6 mutants are temperature sensitive for viability, and accumulate mitochondrial precursor proteins at the restrictive temperature. We show that mas6 does not correspond to any of the presently identified import mutants, and we find that mitochondria isolated from mas6 mutants are defective at an early stage of the mitochondrial protein import pathway. MAS6 encodes a 23-kD protein that contains several potential membrane spanning domains, and yeast strains disrupted for MAS6 are inviable at all temperatures and on all carbon sources. The Mas6 protein is located in the mitochondrial inner membrane and cannot be extracted from the membrane by alkali treatment. Antibodies to the Mas6 protein inhibit import into isolated mitochondria, but only when the outer membrane has been disrupted by osmotic shock. Mas6p therefore represents an essential import component located in the mitochondrial inner membrane.     

The protein import and assembly machinery of the mitochondrial outer membrane

The process of mitochondrial protein import has been studied for many years. Despite this attention, many processes associated with mitochondrial biogenesis are poorly understood. Insight into one of these processes, assembly of beta-barrel proteins into the mitochondrial outer membrane, will be discussed. This review focuses on recent data that suggest that assembly of beta-barrel proteins into the outer mitochondrial membrane is dependent on a newly identified protein complex termed the sorting and assembly machinery (SAM complex). Members of the SAM complex have been identified in both eukaryotic and prokaryotic organisms, suggesting that the process of beta-barrel assembly into membranes has been conserved through evolution.     

Multistep assembly of the protein import channel of the mitochondrial outer membrane

Proteins targeted to mitochondria are transported into the organelle through a high molecular weight complex called the translocase of the outer mitochondrial membrane (TOM). At the core of this machinery is a multisubunit general import pore (GIP) of 400 kDa. Here we report the assembly of the yeast GIP that involves two successive intermediates of 250 kDa and 100 kDa. The precursor of the channel-lining Tom40 is first targeted to the membrane via the receptor proteins Tom20 and Tom22; it then assembles with Tom5 to form the 250 kDa intermediate exposed to the intermembrane space. The 250 kDa intermediate is followed by the formation of the 100 kDa intermediate that associates with Tom6. Maturation to the 400 kDa complex occurs by association of Tom7 and Tom22. Tom7 functions by promoting both the dissociation of the 400 kDa complex and the transition from the 100 kDa intermediate to the mature complex. These results indicate that the dynamic conversion between the 400 kDa complex and the 100 kDa late intermediate allows integration of new precursor subunits into pre-existing complexes.     

A novel import route for an N-anchor mitochondrial outer membrane protein aided by the TIM23 complex

The membrane topology of Om45 in the yeast mitochondrial outer membrane (OM) is under debate. Here, we confirm that Om45 is anchored to the OM from the intermembrane space (IMS) by its N-terminal hydrophobic segment. We show that import of Om45 requires the presequence receptors, Tom20 and Tom22, and the import channel of Tom40. Unlike any of the known OM proteins, Om45 import requires the TIM23 complex in the inner membrane, a translocator for presequence-containing proteins, and the membrane potential (ΔΨ). Therefore, Om45 is anchored to the OM via the IMS by a novel import pathway involving the TIM23 complex.     

Characterization of a human import component of the mitochondrial outer membrane,TOMM70A

Functional mitochondria require up to 1000 proteins to function properly, with 99% synthesized as precursors in the cytoplasm and transported into the mitochondria with the aid of cytosolic chaperones and mitochondrial translocators (import components). Proteins to be imported are chaperoned to the mitochondria by the cytosolic heat shock protein (cHSP70) and are immediately pursued by Translocators of the Outer Membrane (TOMs), followed by transient interactions of the unfolded proteins with Translocators of the Inner Membrane (TIMs). In the present study, we describe a human gene, TOMM70A, orthologous to the yeast Tom70 import component. TOMM70A is ubiquitously expressed in human tissues, maps on chromosome 3q13.1-q13.2 and consists of 12 coding exons spanning over 37 kb. TOMM70A localizes in the mitochondria of COS-7 cells, and in organello import assays confirmed its presence in the Outer Mitochondrial membrane (OM) of rat liver mitochondria. TOMM70A could play a significant role in the import of nuclear-encoded mitochondrial proteins with internal targeting sites such as ADP/ATP carriers and the uncoupling proteins.     

TbLOK1/ATOM19 is a novel subunit of the noncanonical mitochondrial outer membrane protein translocase of Trypanosoma brucei

TbLOK1 has previously been characterized as a trypanosomatid‐specific mitochondrial outer membrane protein whose ablation caused a collapse of the mitochondrial network, disruption of the membrane potential and loss of mitochondrial DNA. Here we show that ablation of TbLOK1 primarily abolishes mitochondrial protein import, both in vivo and in vitro. Co‐immunprecipitations together with blue native gel analysis demonstrate that TbLOK1 is a stable and stoichiometric component of the archaic protein translocase of the outer membrane (ATOM), the highly diverged functional analogue of the TOM complex in other organisms. Furthermore, we show that TbLOK1 together with the other ATOM subunits forms a complex functional network where ablation of individual subunits either causes degradation of a specific set of other subunits or their exclusion from the ATOM complex. In summary these results establish that TbLOK1 is an essential novel subunit of the ATOM complex and thus that its primary molecular function is linked to mitochondrial protein import across the outer membrane. The previously described phenotypes can all be explained as consequences of the lack of mitochondrial protein import. We therefore suggest that in line with the nomenclature of the ATOM complex subunits, TbLOK1 should be renamed to ATOM19.     

The mitochondrial import protein Mim1 promotes biogenesis of multispanning outer membrane proteins

The mitochondrial outer membrane contains translocase complexes for the import of precursor proteins. The translocase of the outer membrane complex functions as a general preprotein entry gate, whereas the sorting and assembly machinery complex mediates membrane insertion of β-barrel proteins of the outer membrane. Several α-helical outer membrane proteins are known to carry multiple transmembrane segments; however, only limited information is available on the biogenesis of these proteins. We report that mitochondria lacking the mitochondrial import protein 1 (Mim1) are impaired in the biogenesis of multispanning outer membrane proteins, whereas overexpression of Mim1 stimulates their import. The Mim1 complex cooperates with the receptor Tom70 in binding of precursor proteins and promotes their insertion and assembly into the outer membrane. We conclude that the Mim1 complex plays a central role in the import of α-helical outer membrane proteins with multiple transmembrane segments.     

A component of the chloroplastic protein import apparatus is targeted to the outer envelope membrane via a novel pathway.

A chloroplastic outer envelope membrane protein of 75 kDa (OEP75) was identified previously as a component of the protein import machinery. Here we provide additional evidence that OEP75 is a component of protein import, present the isolation of a cDNA clone encoding this protein, briefly describe its developmental expression and tissue specificity, and characterize its insertion into the outer envelope membrane. OEP75 was synthesized as a higher molecular weight precursor (prOEP75) which bound to isolated chloroplasts in an in vitro import assay and subsequently was processed to the mature form (mOEP75). During this import assay, two proteins intermediate in size between prOEP75 and mOEP75 were detected. One of these intermediates was also detected in chloroplast envelopes isolated from young pea leaves. Binding and processing of prOEP75 required ATP and one or more surface-exposed proteinaceous components, and was competed by prSSU, a stromal-targeted protein. We propose that the N-terminus of the prOEP75 transit peptide acts as a stromal-targeting domain and a central, hydrophobic region of this transit peptide acts as a stop-transfer domain. A complex route of insertion and processing of prOEP75 may exist to ensure high fidelity targeting of this import component.     

Precise determination of the mitochondrial import signal contained in a 70 kDa protein of yeast mitochondrial outer membrane

A major 70 kDa protein of the yeast mitochondrial outer membrane is coded by a nuclear gene, synthesized on cytoplasmic ribosomes, and transported to the mitochondrial outer membrane. In order to investigate in detail the information necessary for localizing the 70 kDa protein at the outer membrane, we have examined the intracellular and intramitochondrial location of fusion proteins which consist of various lengths of the amino-terminal region of the 70 kDa protein with an enzymatically active beta-galactosidase. The results indicate that the extreme amino-terminal 12 amino acids of the 70 kDa protein function as a targeting sequence, whereas the subsequent uncharged region (up to residue 29) is necessary for "stop-transfer" and "anchoring" functions. Moreover, we have found that a fusion protein which contained the amino-terminal 19 amino acids of the 70 kDa protein is localized on the outer membrane as well as in the matrix space. Changes in the dual localization of this fusion protein accompanied its overproduction or expression in a respiration-deficient yeast mutant.     

An essential role of Sam50 in the protein sorting and assembly machinery of the mitochondrial outer membrane

The preprotein translocase of the outer mitochondrial membrane (TOM complex) contains one essential subunit, the channel Tom40. The assembly pathway of the precursor of Tom40 involves the TOM complex and the sorting and assembly machinery (SAM complex) with the non-essential subunit Mas37. We have identified Sam50, the second essential protein of the mitochondrial outer membrane. Sam50 contains a beta-barrel domain conserved from bacteria to man and is a subunit of the SAM complex. Yeast mutants of Sam50 are defective in the assembly pathways of Tom40 and the abundant outer membrane protein porin, while the import of matrix proteins is not affected. Thus the protein sorting and assembly machinery of the mitochondrial outer membrane involves an essential, conserved protein.     

The AAA-ATPase p97 is essential for outer mitochondrial membrane protein turnover

Recent studies have revealed a role for the ubiquitin/proteasome system in the regulation and turnover of outer mitochondrial membrane (OMM)-associated proteins. Although several molecular components required for this process have been identified, the mechanism of proteasome-dependent degradation of OMM-associated proteins is currently unclear. We show that an AAA-ATPase, p97, is required for the proteasomal degradation of Mcl1 and Mfn1, two unrelated OMM proteins with short half-lives. A number of biochemical assays, as well as imaging of changes in localization of photoactivable GFP-fused Mcl1, revealed that p97 regulates the retrotranslocation of Mcl1 from mitochondria to the cytosol, prior to, or concurrent with, proteasomal degradation. Mcl1 retrotranslocation from the OMM depends on the activity of the ATPase domain of p97. Furthermore, p97-mediated retrotranslocation of Mcl1 can be recapitulated in vitro, confirming a direct mitochondrial role for p97. Our results establish p97 as a novel and essential component of the OMM-associated protein degradation pathway.     

Two novel proteins in the mitochondrial outer membrane mediate beta-barrel protein assembly

Mitochondrial outer and inner membranes contain translocators that achieve protein translocation across and/or insertion into the membranes. Recent evidence has shown that mitochondrial beta-barrel protein assembly in the outer membrane requires specific translocator proteins in addition to the components of the general translocator complex in the outer membrane, the TOM40 complex. Here we report two novel mitochondrial outer membrane proteins in yeast, Tom13 and Tom38/Sam35, that mediate assembly of mitochondrial beta-barrel proteins, Tom40, and/or porin in the outer membrane. Depletion of Tom13 or Tom38/Sam35 affects assembly pathways of the beta-barrel proteins differently, suggesting that they mediate different steps of the complex assembly processes of beta-barrel proteins in the outer membrane.     

Characterization of the novel mitochondrial protein import component, Tom34, in mammalian cells

Tom34 is a newly-found component of the mitochondrial protein import machinery in mammalian cells with no apparent counterpart in fungi. RNA blot and immunoblot analyses showed that the expression of Tom34 varies among tissues and differs from that of the core translocase component Tom20. In contrast to a previous report [Nuttal, S.D. et al. (1997) DNA Cell Biol. 16, 1067-1074], the present study using a newly-prepared anti-Tom34 antibody with a high titer showed that Tom34 is present largely in the cytosolic fraction and partly in the mitochondrial and membrane fractions after fractionation of tissues and cells, and that the membrane-associated form is largely extractable with 0.1 M sodium carbonate. The in vitro import of preproteins into isolated rat mitochondria was strongly inhibited by DeltahTom34 which lacks the NH2-terminal hydrophobic region of human Tom34 (hTom34). Import was also strongly inhibited by anti-hTom34. In pulse-chase experiments using COS-7 cells, pre-ornithine transcarbamylase (pOTC) was rapidly processed to the mature form. Coexpression of hTom34 resulted in a stimulation of pOTC processing, whereas the coexpression of hTom34 antisense RNA caused inhibition. The results confirm that Tom34 plays a role in mitochondrial protein import in mammals, and suggest it to be an ancillary component of the translocation machinery in mammalian cells.     

Cytosolic factor- and TOM-independent import of C-tail-anchored mitochondrial outer membrane proteins

C-tail-anchored (C-TA) proteins are anchored to specific organelle membranes by a single transmembrane segment (TMS) at the C-terminus, extruding the N-terminal functional domains into the cytoplasm in which the TMS and following basic segment function as the membrane-targeting signals. Here, we analyzed the import route of mitochondrial outer membrane (MOM) C-TA proteins, Bak, Bcl-XL, and Omp25, using digitonin-permeabilized HeLa cells, which provide specific and efficient import under competitive conditions. These experiments revealed that (i) C-TA proteins were imported to the MOM through a common pathway independent of the components of the preprotein translocase of the outer membrane, (ii) the C-TA protein-targeting signal functioned autonomously in the absence of cytoplasmic factors that specifically recognize the targeting signals and deliver the preproteins to the MOM, (iii) the function of a cytoplasmic chaperone was required if the cytoplasmic domains of the C-TA proteins assumed an import-incompetent conformation, and intriguingly, (iv) the MOM-targeting signal of Bak, in the context of the Bak molecule, required activation by the interaction of its cytoplasmic domain with VDAC2 before MOM targeting.     

Multispan mitochondrial outer membrane protein Ugo1 follows a unique Mim1-dependent import pathway

The mitochondrial outer membrane (MOM) harbors several multispan proteins that execute various functions. Despite their importance, the mechanisms by which these proteins are recognized and inserted into the outer membrane remain largely unclear. In this paper, we address this issue using yeast mitochondria and the multispan protein Ugo1. Using a specific insertion assay and analysis by native gel electrophoresis, we show that the import receptor Tom70, but not its partner Tom20, is involved in the initial recognition of the Ugo1 precursor. Surprisingly, the import pore formed by the translocase of the outer membrane complex appears not to be required for the insertion process. Conversely, the multifunctional outer membrane protein mitochondrial import 1 (Mim1) plays a central role in mediating the insertion of Ugo1. Collectively, these results suggest that Ugo1 is inserted into the MOM by a novel pathway in which Tom70 and Mim1 contribute to the efficiency and selectivity of the process.     

The transport machinery for the import of preproteins across the outer mitochondrial membrane

In order for proteins to be imported into subcellular compartments, they must first traverse the organellar membranes. In mitochondria, hydrophilic protein channels in both the outer and inner membranes serve such a purpose. Recently, the channel protein of the outer mitochondrial membrane was identified to be Tom40. Tom40 is found in a high molecular weight complex termed the general import pore (GIP) complex where it is tightly associated with the receptor protein Tom22 along with Tom7, Tom6 and Tom5. Tom7 and Tom6 seem to modulate the dynamics of the GIP complex while Tom5 is involved in preprotein transfer from receptors to Tom40. The receptor proteins Tom70 and Tom20 associate with this complex in a weaker manner where they are involved in the initial recognition of preproteins. This review focuses on the identification and characterisation of the transport machinery of the outer mitochondrial membrane and how they are involved in the co-ordination and regulation of events required for the translocation of preproteins into mitochondria.     

A plant outer mitochondrial membrane protein with high amino acid sequence identity to a chloroplast protein import receptor

The conductivity of two photosynthetic protein–pigment complexes, a light harvesting 2 complex and a reaction center, was measured with an atomic force microscope capable of performing electrical measurements. Current–voltage measurements were performed on complexes embedded in their natural environment. Embedding the complexes in lipid bilayers made it possible to discuss the different conduction behaviors of the two complexes in light of their atomic structure.