Protein Translocation Machinery Research Articles

New insights into the structure and dynamics of the TOM complex in mitochondria.

To date, there is no general physical model of the mechanism by which unfolded polypeptide chains with different properties are imported into the mitochondria. At the molecular level, it is still unclear how transit polypeptides approach, are captured by the protein translocation machinery in the outer mitochondrial membrane, and how they subsequently cross the entropic barrier of a protein translocation pore to enter the intermembrane space. This deficiency has been due to the lack of detailed structural and dynamic information about the membrane pores. In this review, we focus on the recently determined sub-nanometer cryo-EM structures and our current knowledge of the dynamics of the mitochondrial two-pore outer membrane protein translocation machinery (TOM core complex), which provide a starting point for addressing the above questions. Of particular interest are recent discoveries showing that the TOM core complex can act as a mechanosensor, where the pores close as a result of interaction with membrane-proximal structures. We highlight unusual and new correlations between the structural elements of the TOM complexes and their dynamic behavior in the membrane environment.

The peroxisome protein translocation machinery is developmentally regulated in the fungus Podospora anserina.

Sexual reproduction allows eukaryotic organisms to produce genetically diverse progeny. This process relies on meiosis, a reductional division that enables ploidy maintenance and genetic recombination. Meiotic differentiation also involves the renewal of cell functioning to promote offspring rejuvenation. Research in the model fungus Podospora anserina has shown that this process involves a complex regulation of the function and dynamics of different organelles, including peroxisomes. These organelles are critical for meiosis induction and play further significant roles in meiotic development. Here we show that PEX13-a key constituent of the protein conduit through which the proteins defining peroxisome function reach into the organelle-is subject to a developmental regulation that almost certainly involves its selective ubiquitination-dependent removal and that modulates its abundance throughout meiotic development and at different sexual differentiation processes. Our results show that meiotic development involves a complex developmental regulation of the peroxisome protein translocation system.

Role of Hsp70 in Post-Translational Protein Targeting: Tail-Anchored Membrane Proteins and Beyond.

The Hsp70 family of molecular chaperones acts as a central 'hub' in the cell that interacts with numerous newly synthesized proteins to assist in their biogenesis. Apart from its central and well-established role in facilitating protein folding, Hsp70s also act as key decision points in the cellular chaperone network that direct client proteins to distinct biogenesis and quality control pathways. In this paper, we review accumulating data that illustrate a new branch in the Hsp70 network: the post-translational targeting of nascent membrane and organellar proteins to diverse cellular organelles. Work in multiple pathways suggests that Hsp70, via its ability to interact with components of protein targeting and translocation machineries, can initiate elaborate substrate relays in a sophisticated cascade of chaperones, cochaperones, and receptor proteins, and thus provide a mechanism to safeguard and deliver nascent membrane proteins to the correct cellular membrane. We discuss the mechanistic principles gleaned from better-studied Hsp70-dependent targeting pathways and outline the observations and outstanding questions in less well-studied systems.

OAB-028: FAM46C-dependent tuning of endoplasmic reticulum capacity in Multiple Myeloma

<h3>Background</h3> Protein secretion is a driving force in evolution and tissue specialization. Plasma cells (PC), responsible for intense antibody (Ab) production, are paradigmatic professional secretors whose differentiation entails rapid endoplasmic reticulum (ER) expansion and concerted expression of the protein translocation, folding and trafficking machinery. A novel player in PC differentiation, recently implicated in Ab immunity is the non-canonical poly(A) polymerase FAM46C/TENT5C. <h3>Methods</h3> In developing PCs, FAM46C is induced to polyadenylate and enhance the translation of a number of mRNAs encoding Igs and ER-targeted proteins [1,2]. Being deleted or mutated in up to 20% multiple myeloma (MM) patients, but intact in other cancers, the FAM46C gene is among the most frequent genomic hits in MM and a myeloma-specific onco-suppressor [3,4]. <h3>Results</h3> We have recently shown that the ER-specific action of FAM46C depends on its interaction with the ER transmembrane FNDC3 proteins. As a result, FAM46C concertedly boosts the expression of ER and Golgi proteins, potently upsizing the secretory apparatus and Ig secretion [5]. Our data suggest that in MM the FAM46C gene is under selective pressure to contain the proteosynthetic, oxidative and metabolic stress associated with Ig secretion, favoring myeloma cell survival and growth. In keeping with this, exogenous re-expression of FAM46C in mutated MM cells raised Ig secretory capacity beyond sustainability, causing ATP shortage, ROS accumulation and apoptosis. Interestingly, although able to increase the secretory capacity across different cell types, FAM46C overexpression induced apoptosis exclusively in MM cells, being well tolerated in non-professional secretors, suggesting a key role of the secretory cargo in FAM46C toxicity. Moreover, we discovered a broader function of FAM46C in ER homeostasis beyond mRNA stabilization. Indeed, we found that FAM46C is part of a novel integrated network coordinating the biogenesis of ribonucleoprotein complexes, ER protein translation and import, and ER expansion, to harmonize massive Ab production with protein homeostasis. Finally, our proteomic analysis unveiled a surprising opposite modulation of signal recognition particle (SRP) proteins mediated by FAM46C. Indeed, while 4 out of 6 SRP proteins were positively regulated by FAM46C, the heterodimeric members SRP9 and SRP14, mediating translational arrest, increased upon FAM46C loss. <h3>Conclusions</h3> We infer that PCs have the ability to sense FAM46C-induced ER expansion and modulate SRP composition to minimize translational pausing and maximize Ab manufacture. This novel integrated network offers a framework to identify unprecedented highly specific therapeutic targets against PC dyscrasias.

Accessing Mitochondrial Protein Import in Living Cells by Protein Microinjection.

Mitochondrial protein biogenesis relies almost exclusively on the expression of nuclear-encoded polypeptides. The current model postulates that most of these proteins have to be delivered to their final mitochondrial destination after their synthesis in the cytoplasm. However, the knowledge of this process remains limited due to the absence of proper experimental real-time approaches to study mitochondria in their native cellular environment. We developed a gentle microinjection procedure for fluorescent reporter proteins allowing a direct non-invasive study of protein transport in living cells. As a proof of principle, we visualized potential-dependent protein import into mitochondria inside intact cells in real-time. We validated that our approach does not distort mitochondrial morphology and preserves the endogenous expression system as well as mitochondrial protein translocation machinery. We observed that a release of nascent polypeptides chains from actively translating cellular ribosomes by puromycin strongly increased the import rate of the microinjected pre-protein. This suggests that a substantial amount of mitochondrial translocase complexes was involved in co-translational protein import of endogenously expressed pre-proteins. Our protein microinjection method opens new possibilities to study the role of mitochondrial protein import in cell models of various pathological conditions as well as aging processes.

The Carbapenemase BKC-1 from Klebsiella pneumoniae Is Adapted for Translocation by Both the Tat and Sec Translocons.

ABSTRACTThe cell envelope of Gram-negative bacteria consists of two membranes surrounding the periplasm and peptidoglycan layer. β-Lactam antibiotics target the periplasmic penicillin-binding proteins that synthesize peptidoglycan, resulting in cell death. The primary means by which bacterial species resist the effects of β-lactam drugs is to populate the periplasmic space with β-lactamases. Resistance to β-lactam drugs is spread by lateral transfer of genes encoding β-lactamases from one species of bacteria to another. However, the resistance phenotype depends in turn on these “alien” protein sequences being recognized and exported across the cytoplasmic membrane by either the Sec or Tat protein translocation machinery of the new bacterial host. Here, we examine BKC-1, a carbapenemase from an unknown bacterial source that has been identified in a single clinical isolate of Klebsiella pneumoniae. BKC-1 was shown to be located in the periplasm, and functional in both K. pneumoniae and Escherichia coli. Sequence analysis revealed the presence of an unusual signal peptide with a twin arginine motif and a duplicated hydrophobic region. Biochemical assays showed this signal peptide directs BKC-1 for translocation by both Sec and Tat translocons. This is one of the few descriptions of a periplasmic protein that is functionally translocated by both export pathways in the same organism, and we suggest it represents a snapshot of evolution for a β-lactamase adapting to functionality in a new host.

Mitochondrial protein import dysfunction: mitochondrial disease, neurodegenerative disease and cancer.

The majority of proteins localised to mitochondria are encoded by the nuclear genome, with approximately 1500 proteins imported into mammalian mitochondria. Dysfunction in this fundamental cellular process is linked to a variety of pathologies including neuropathies, cardiovascular disorders, myopathies, neurodegenerative diseases and cancer, demonstrating the importance of mitochondrial protein import machinery for cellular function. Correct import of proteins into mitochondria requires the co-ordinated activity of multimeric protein translocation and sorting machineries located in both the outer and inner mitochondrial membranes, directing the imported proteins to the destined mitochondrial compartment. This dynamic process maintains cellular homeostasis, and its dysregulation significantly affects cellular signalling pathways and metabolism. This review summarises current knowledge of the mammalian mitochondrial import machinery and the pathological consequences of mutation of its components. In addition, we will discuss the role of mitochondrial import in cancer, and our current understanding of the role of mitochondrial import in neurodegenerative diseases including Alzheimer's disease, Huntington's disease and Parkinson's disease.

Structural snapshots of the cellular folded protein translocation machinery Bcs1.

Proteins destined to various intra- and extra-cellular locations must traverse membranes most frequently in an unfolded form. When the proteins being translocated need to remain in a folded state, specialized cellular transport machinery is used. One such machine is the membrane-bound AAA protein Bcs1 (Bcs1), which assists the iron-sulfur protein, an essential subunit of the respiratory Complex III, across the mitochondrial inner membrane. Recent structure determinations of mouse and yeast Bcs1 in three different nucleotide states reveal its homo-heptameric association and at least two dramatically different conformations. The apo and ADP-bound structures are similar, both containing a large substrate-binding cavity accessible to the mitochondrial matrix space, which contracts by concerted motion of the ATPase domains upon ATP binding, suggesting that bound substrate could then be pushed across the membrane. ATP hydrolysis drives substrate release and resets Bcs1 conformation back to the apo/ADP form. These structures shed new light on the mechanism of folded protein translocation across a membrane, provide better understanding on the assembly process of the respiratory Complex III, and correlate clinical presentations of disease-associated mutations with their locations in the 3D structure.

Escherichia coli Can Adapt Its Protein Translocation Machinery for Enhanced Periplasmic Recombinant Protein Production

Recently, we engineered a tunable rhamnose promoter-based setup for the production of recombinant proteins in E. coli. This setup enabled us to show that being able to precisely set the production rate of a secretory recombinant protein is critical to enhance protein production yields in the periplasm. It is assumed that precisely setting the production rate of a secretory recombinant protein is required to harmonize its production rate with the protein translocation capacity of the cell. Here, using proteome analysis we show that enhancing periplasmic production of human Growth Hormone (hGH) using the tunable rhamnose promoter-based setup is accompanied by increased accumulation levels of at least three key players in protein translocation; the peripheral motor of the Sec-translocon (SecA), leader peptidase (LepB), and the cytoplasmic membrane protein integrase/chaperone (YidC). Thus, enhancing periplasmic hGH production leads to increased Sec-translocon capacity, increased capacity to cleave signal peptides from secretory proteins and an increased capacity of an alternative membrane protein biogenesis pathway, which frees up Sec-translocon capacity for protein secretion. When cells with enhanced periplasmic hGH production yields were harvested and subsequently cultured in the absence of inducer, SecA, LepB, and YidC levels went down again. This indicates that when using the tunable rhamnose-promoter system to enhance the production of a protein in the periplasm, E. coli can adapt its protein translocation machinery for enhanced recombinant protein production in the periplasm.

Membrane proteomic analysis reveals overlapping and independent functions of Streptococcus mutans Ffh, YidC1, and YidC2.

A comparative proteomic analysis was utilized to evaluate similarities and differences in membrane samples derived from the cariogenic bacterium Streptococcus mutans, including the wild-type strain and four mutants devoid of protein translocation machinery components, specifically ∆ffh, ∆yidC1, ∆yidC2, or ∆ffh/yidC1. The purpose of this work was to determine the extent to which the encoded proteins operate individually or in concert with one another and to identify the potential substrates of the respective pathways. Ffh is the principal protein component of the signal recognition particle (SRP), while yidC1 and yidC2 are dual paralogs encoding members of the YidC/Oxa/Alb family of membrane-localized chaperone insertases. Our results suggest that the co-translational SRP pathway works in concert with either YidC1 or YidC2 specifically, or with no preference for paralog, in the insertion of most membrane-localized substrates. A few instances were identified in which the SRP pathway alone, or one of the YidCs alone, appeared to be most relevant. These data shed light on underlying reasons for differing phenotypic consequences of ffh, yidC1 or yidC2 deletion. Our data further suggest that many membrane proteins present in a ∆yidC2 background may be non-functional, that ∆yidC1 is better able to adapt physiologically to the loss of this paralog, that shared phenotypic properties of ∆ffh and ∆yidC2 mutants can stem from impacts on different proteins, and that independent binding to ribosomal proteins is not a primary functional activity of YidC2. Lastly, genomic mutations accumulate in a ∆yidC2 background coincident with phenotypic reversion, including an apparent W138R suppressor mutation within yidC1.

Interactions of protein translocation machinery components of Streptococcus mutans

BackgroundPivotal virulence properties of the cariogenic bacterium Streptococcus mutans depend on secreted and integral membrane proteins. Bacterial protein trafficking involves the signal recognition particle (SRP) pathway components Ffh, scRNA and FtsY, the SecYEG translocon, the SecA motor protein, and membrane‐localized YidC chaperone/insertases. Unlike Escherichia coli, S. mutans survives loss of the SRP pathway. In addition, S. mutans has two yidC paralogs. The phenotype of a DyidC2 mutant largely parallels that of Dffh, while that of DyidC1 is far less severe. Also in S. mutans, YlxM acts as an accessory factor of the SRP that modulates GTP hydrolysis activities of Ffh and FtsY. The goal of this study is to identify interactions among S. mutans transport machinery components to better understand the respective functions of YidC1 and YidC2 alone and in concert with the SRP, ribosome, and/or Sec translocon that can affect the destiny of translocated proteins.MethodsA chemical cross‐linking approach was employed, whereby whole cell lysates (wcl) were treated with formaldehyde followed by Western blotting using anti‐Ffh, FtsY, YidC1 and YidC2 antibodies and mass spectrometry (MS) analysis of corresponding gel‐shifted bands. Wcl from WT and DyidC2 strains were also reacted with a‐YidC2 antibodies coupled to magnetic Dynabeads™ and co‐captured proteins identified by MS. In addition, biolayer interferometry (BLI) was used to evaluate specific interactions of the C‐terminal tails of YidC1 and YidC2, engineered as glutathione‐S‐transferase fusion proteins (GST‐C1 and GST‐C2), with the SRP components Ffh and YlxM. The same GST‐constructs were also subjected to 2D Difference Gel Electrophoresis (DIGE), in which each was reacted with wcl, captured with glutathione affinity resin, and labelled individually with fluorescent dyes. Spots unique to the GST‐C1 and/or GST‐C2 samples were analysed by MS.Results and ConclusionBand‐shift patterns detected with anti‐Ffh, FtsY and YidC2 antisera were similar to one another and distinct from that of anti‐YidC1. Cross‐linked proteins included elongation factor Tu, DNA‐directed RNA polymerase subunit b, and HSP 70. Immunocaptured proteins from anti‐YidC2 Dynabeads™ included two small, transmembrane proteins, LemA and SMU_1719c, which may represent translocation substrates. Both GST‐C1 and GST‐C2 interacted with Ffh by BLI with binding influenced by YlxM. Over one hundred spots were identified by DIGE. Among differentially identified proteins in the GST‐C1 sample were DnaK, RNA polymerase subunit d, and cell division protein FtsH. Ribosomal proteins S7 and L13 were prevalent in spots of the GST‐C2 sample, whereas ribosomal protein L2, DNA polymerase III g/t subunit, and the Cas5 homolog were all more abundant in spots from GST‐C1 and GST‐C2 samples compared to GST. This information suggests a coupled cellular machinery that integrates transcription and translation with protein transport and folding.Support or Funding InformationNIH R01DE008007This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Structure of the post-translational protein translocation machinery of the ER membrane.

Many proteins must translocate through the protein-conducting Sec61 channel in the eukaryotic endoplasmic reticulum membrane or the SecY channel in the prokaryotic plasma membrane1,2. Proteins with hydrophobic signal sequences are first recognized by the signal recognition particle (SRP)3,4 and then moved co-translationally through the Sec61/SecY channel by the associated translating ribosome. Substrates with less hydrophobic signal sequences bypass SRP and are moved through the channel post-translationally5,6. In eukaryotic cells, post-translational translocation is mediated by the association of the Sec61 channel with another membrane protein complex, the Sec62/Sec63 complex7–9, and substrates are moved through the channel by the luminal BiP ATPase9. How the Sec62/63 complex activates the Sec61 channel for post-translational translocation is unclear. Here, we report the electron cryo-microscopy (cryo-EM) structure of the Sec complex from S. cerevisiae, consisting of the Sec61 channel and the Sec62, Sec63, Sec71, and Sec72 proteins. Sec63 causes wide opening of the lateral gate of the Sec61 channel, priming it for the passage of low-hydrophobicity signal sequences into the lipid phase, without displacing the channel’s plug domain. Lateral channel opening is triggered by Sec63 interacting with both cytosolic loops in the C-terminal half of Sec61 and trans-membrane (TM) segments in the N-terminal half of the Sec61 channel. The cytosolic Brl domain of Sec63 blocks ribosome binding to the channel and recruits Sec71 and Sec72, positioning them for the capture of polypeptides associated with cytosolic Hsp70 (ref.10). Our structure shows how the Sec61 channel is activated for post-translational protein translocation.

The unfolded protein response and endoplasmic reticulum protein targeting machineries converge on the stress sensor IRE1.

The protein folding capacity of the endoplasmic reticulum (ER) is tightly regulated by a network of signaling pathways, known as the unfolded protein response (UPR). UPR sensors monitor the ER folding status to adjust ER folding capacity according to need. To understand how the UPR sensor IRE1 maintains ER homeostasis, we identified zero-length crosslinks of RNA to IRE1 with single nucleotide precision in vivo. We found that IRE1 specifically crosslinks to a subset of ER-targeted mRNAs, SRP RNA, ribosomal and transfer RNAs. Crosslink sites cluster in a discrete region of the ribosome surface spanning from the A-site to the polypeptide exit tunnel. Moreover, IRE1 binds to purified 80S ribosomes with high affinity, indicating association with ER-bound ribosomes. Our results suggest that the ER protein translocation and targeting machineries work together with the UPR to tune the ER's protein folding load.

The intrinsically disordered nature of the peroxisomal protein translocation machinery.

Despite having a membrane that is impermeable to all but the smallest of metabolites, peroxisomes acquire their newly synthesized (cytosolic) matrix proteins in an already folded conformation. In some cases, even oligomeric proteins have been reported to translocate the organelle membrane. The protein sorting machinery that accomplishes this feat must be rather flexible and, unsurprisingly, several of its key components have large intrinsically disordered domains. Here, we provide an overview on these domains and their interactions trying to infer their functional roles in this protein sorting pathway.

Sec61 blockade by mycolactone: A central mechanism in Buruli ulcer disease.

Infection with Mycobacterium ulcerans results in a necrotising skin disease known as a Buruli ulcer, the pathology of which is directly linked to the bacterial production of the toxin mycolactone. Recent studies have identified the protein translocation machinery of the endoplasmic reticulum (ER) membrane as the primary cellular target of mycolactone, and shown that the toxin binds to the core subunit of the Sec61 complex. Mycolactone binding strongly inhibits the capacity of the Sec61 translocon to transport newly synthesised membrane and secretory proteins into and across the ER membrane. Since the ER acts as the entry point for the mammalian secretory pathway, and hence regulates initial access to the entire endomembrane system, mycolactone-treated cells have a reduced ability to produce a range of proteins including secretory cytokines and plasma membrane receptors. The global effect of this molecular blockade of protein translocation at the ER is that the host is unable to mount an effective immune response to the underlying mycobacterial infection. Prolonged exposure to mycolactone is normally cytotoxic, since it triggers stress responses activating the transcription factor ATF4 and ultimately inducing apoptosis.

New Perspectives on Chloroplast Protein Import.

Virtually all chloroplasts in extant photosynthetic eukaryotes derive from a single endosymbiotic event that probably occurred more than a billion years ago between a host eukaryotic cell and a cyanobacterium-like ancestor. Many endosymbiont genes were subsequently transferred to the host nuclear genome, concomitant with the establishment of a system for protein transport through the chloroplast double-membrane envelope. Presently, 2,000-3,000 different nucleus-encoded chloroplast proteins must be imported into the chloroplast following their synthesis in the cytosol. The TOC (translocon at the outer envelope membrane of chloroplasts) and TIC (translocon at the inner envelope membrane of chloroplasts) complexes are protein translocation machineries at the outer and inner envelope membranes, respectively, that facilitate this chloroplast protein import with the aid of a TIC-associated ATP-driven import motor. All the essential components of this protein import system seemed to have been identified through biochemical analyses and subsequent genetic studies that initiated in the late 1990s. However, in 2013, the Nakai group reported a novel inner envelope membrane TIC complex, for which a novel ATP-driven import motor associated with this TIC complex is likely to exist. In this mini review, I will summarize these recent discoveries together with new, or reanalyzed, data presented by other groups in recent years. Whereas the precise concurrent view of chloroplast protein import is still a matter of some debate, it is anticipated that the entire TOC/TIC/ATP motor system, including any novel components, will be conclusively established in the next decade. Such findings may lead to an extensively revised view of the evolution and molecular mechanisms of chloroplast protein import.

Electric-Field-Induced Protein Translocation via a Conformational Transition in SecDF: An MD Study

SecDF is an important component of the Sec protein translocation machinery embedded in the bacterial membrane, which is associated with many functions, such as stabilizing other Sec translocon components within the membrane, maintaining the transmembrane (TM) potential, and facilitating the ATP-independent stage of the translocation mechanism. Related studies suggest that SecDF undergoes functionally important conformational changes that involve mainly its P1-head domain and that these changes are coupled with the proton motive force (Δp). However, there still is not a clear understanding of how SecDF functions, its exact role in the translocation machinery, and how its function is related to Δp. Here, using all-atom molecular dynamics simulations combined with umbrella sampling, we study the P1-head conformational change and how it is coupled to the proton motive force. We report potentials of mean force along a root-mean-square-distance-based reaction coordinate obtained in the presence and absence of the TM electrical potential. Our results show that the interaction of the P1 domain dipole moment with the TM electrical field considerably lowers the free-energy barrier in the direction of F-form to I-form transition.

Ubiquitin-Proteasome-Dependent Regulation of Bidirectional Communication between Plastids and the Nucleus.

Plastids are DNA-containing organelles and can have unique differentiation states depending on age, tissue, and environment. Plastid biogenesis is optimized by bidirectional communication between plastids and the nucleus. Import of nuclear-encoded proteins into plastids serves as anterograde signals and vice versa, plastids themselves send retrograde signals to the nucleus, thereby controlling de novo synthesis of nuclear-encoded plastid proteins. Recently, it has become increasingly evident that the ubiquitin–proteasome system regulates both the import of anterograde plastid proteins and retrograde signaling from plastids to the nucleus. Targets of ubiquitin–proteasome regulation include unimported chloroplast precursor proteins in the cytosol, protein translocation machinery at the chloroplast surface, and transcription factors in the nucleus. This review will focus on the mechanism through which the ubiquitin–proteasome system optimizes plastid biogenesis and plant development through the regulation of nuclear–plastid interactions.

Dynamic Lipid-dependent Modulation of Protein Topology by Post-translational Phosphorylation

Membrane protein topology and folding are governed by structural principles and topogenic signals that are recognized and decoded by the protein insertion and translocation machineries at the time of initial membrane insertion and folding. We previously demonstrated that the lipid environment is also a determinant of initial protein topology, which is dynamically responsive to post-assembly changes in membrane lipid composition. However, the effect on protein topology of post-assembly phosphorylation of amino acids localized within initially cytoplasmically oriented extramembrane domains has never been investigated. Here, we show in a controlled in vitro system that phosphorylation of a membrane protein can trigger a change in topological arrangement. The rate of change occurred on a scale of seconds, comparable with the rates observed upon changes in the protein lipid environment. The rate and extent of topological rearrangement were dependent on the charges of extramembrane domains and the lipid bilayer surface. Using model membranes mimicking the lipid compositions of eukaryotic organelles, we determined that anionic lipids, cholesterol, sphingomyelin, and membrane fluidity play critical roles in these processes. Our results demonstrate how post-translational modifications may influence membrane protein topology in a lipid-dependent manner, both along the organelle trafficking pathway and at their final destination. The results provide further evidence that membrane protein topology is dynamic, integrating for the first time the effect of changes in lipid composition and regulators of cellular processes. The discovery of a new topology regulatory mechanism opens additional avenues for understanding unexplored structure-function relationships and the development of optimized topology prediction tools.

The non-canonical mitochondrial inner membrane presequence translocase of trypanosomatids contains two essential rhomboid-like proteins.

Mitochondrial protein import is essential for all eukaryotes. Here we show that the early diverging eukaryote Trypanosoma brucei has a non-canonical inner membrane (IM) protein translocation machinery. Besides TbTim17, the single member of the Tim17/22/23 family in trypanosomes, the presequence translocase contains nine subunits that co-purify in reciprocal immunoprecipitations and with a presequence-containing substrate that is trapped in the translocation channel. Two of the newly discovered subunits are rhomboid-like proteins, which are essential for growth and mitochondrial protein import. Rhomboid-like proteins were proposed to form the protein translocation pore of the ER-associated degradation system, suggesting that they may contribute to pore formation in the presequence translocase of T. brucei. Pulldown of import-arrested mitochondrial carrier protein shows that the carrier translocase shares eight subunits with the presequence translocase. This indicates that T. brucei may have a single IM translocase that with compositional variations mediates import of presequence-containing and carrier proteins.