ABSTRACT
The mitochondrial outer membrane harbors two protein translocases that are essential for cell viability: the translocase of the outer mitochondrial membrane (TOM) and the sorting and assembly machinery (SAM). The precursors of ß-barrel proteins use both translocases-TOM for import to the intermembrane space and SAM for export into the outer membrane. It is unknown if the translocases cooperate and where the ß-barrel of newly imported proteins is formed. We established a position-specific assay for monitoring ß-barrel formation in vivo and in organello and demonstrated that the ß-barrel was formed and membrane inserted while the precursor was bound to SAM. ß-barrel formation was inhibited by SAM mutants and, unexpectedly, by mutants of the central import receptor, Tom22. We show that the cytosolic domain of Tom22 links TOM and SAM into a supercomplex, facilitating precursor transfer on the intermembrane space side. Our study reveals receptor-mediated coupling of import and export translocases as a means of precursor channeling.
Subject(s)
Mitochondria/metabolism , Mitochondrial Membrane Transport Proteins/metabolism , Mitochondrial Proteins/metabolism , Protein Transport , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Mitochondrial Membrane Transport Proteins/genetics , Mitochondrial Proteins/chemistry , Mutation , Porins/chemistry , Porins/metabolism , Protein Folding , Protein Structure, Secondary , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/geneticsABSTRACT
Mitochondria import a large number of nuclear-encoded proteins via membrane-bound transport machineries; however, little is known about regulation of the preprotein translocases. We report that the main protein entry gate of mitochondria, the translocase of the outer membrane (TOM complex), is phosphorylated by cytosolic kinases-in particular, casein kinase 2 (CK2) and protein kinase A (PKA). CK2 promotes biogenesis of the TOM complex by phosphorylation of two key components, the receptor Tom22 and the import protein Mim1, which in turn are required for import of further Tom proteins. Inactivation of CK2 decreases the levels of the TOM complex and thus mitochondrial protein import. PKA phosphorylates Tom70 under nonrespiring conditions, thereby inhibiting its receptor activity and the import of mitochondrial metabolite carriers. We conclude that cytosolic kinases exert stimulatory and inhibitory effects on biogenesis and function of the TOM complex and thus regulate protein import into mitochondria.
Subject(s)
Casein Kinase II/metabolism , Cyclic AMP-Dependent Protein Kinases/metabolism , Cytosol/enzymology , Mitochondria/metabolism , Mitochondrial Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Carrier Proteins/metabolism , Cytosol/metabolism , Mitochondrial Membrane Transport Proteins/metabolism , Mitochondrial Precursor Protein Import Complex Proteins , Phosphorylation , Protein Transport , Saccharomyces cerevisiae/cytology , Saccharomyces cerevisiae Proteins/metabolismABSTRACT
Communication of mitochondria with the rest of the cell requires beta-barrel proteins of the outer membrane. All beta-barrel proteins are synthesized as precursors in the cytosol and imported into mitochondria by the general translocase TOM and the sorting machinery SAM. The SAM complex contains two proteins essential for cell viability, the channel-forming Sam50 and Sam35. We have identified the sorting signal of mitochondrial beta-barrel proteins that is universal in all eukaryotic kingdoms. The beta-signal initiates precursor insertion into a hydrophilic, proteinaceous membrane environment by forming a ternary complex with Sam35 and Sam50. Sam35 recognizes the beta-signal, inducing a major conductance increase of the Sam50 channel. Subsequent precursor release from SAM is coupled to integration into the lipid phase. We propose that a two-stage mechanism of signal-driven insertion into a membrane protein complex and subsequent integration into the lipid phase may represent a general mechanism for biogenesis of beta-barrel proteins.
Subject(s)
Mitochondrial Membranes/metabolism , Mitochondrial Proteins/metabolism , Membrane Transport Proteins/chemistry , Membrane Transport Proteins/metabolism , Mitochondria/metabolism , Mitochondrial Membrane Transport Proteins , Mitochondrial Membranes/chemistry , Mitochondrial Proteins/chemistry , Protein Sorting Signals , Protein Structure, Secondary , Protein Structure, Tertiary , Protein Transport , Saccharomyces cerevisiae/cytology , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/metabolismABSTRACT
The majority of preproteins destined for mitochondria carry N-terminal presequences. The presequence translocase of the inner mitochondrial membrane (TIM23 complex) plays a central role in protein sorting. Preproteins are either translocated through the TIM23 complex into the matrix or are laterally released into the inner membrane. We report that the small hydrophobic protein Mgr2 controls the lateral release of preproteins. Mgr2 interacts with preproteins in transit through the TIM23 complex. Overexpression of Mgr2 delays preprotein release, whereas a lack of Mgr2 promotes preprotein sorting into the inner membrane. Preproteins with a defective inner membrane sorting signal are translocated into the matrix in wild-type mitochondria but are released into the inner membrane in Mgr2-deficient mitochondria. We conclude that Mgr2 functions as a lateral gatekeeper of the mitochondrial presequence translocase, providing quality control for the membrane sorting of preproteins.
Subject(s)
Membrane Proteins/metabolism , Membrane Transport Proteins/metabolism , Mitochondrial Membranes/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , GTP-Binding Proteins/metabolism , Membrane Proteins/genetics , Methotrexate/pharmacology , Mitochondria/metabolism , Mitochondria/ultrastructure , Mitochondrial Precursor Protein Import Complex Proteins , Mitochondrial Proteins/metabolism , Protein Transport/drug effects , Saccharomyces cerevisiae Proteins/geneticsABSTRACT
The mitochondrial inner membrane harbors the complexes of the respiratory chain and translocase complexes for precursor proteins. We have identified a further subunit of the carrier translocase (TIM22 complex) that surprisingly is identical to subunit 3 of respiratory complex II, succinate dehydrogenase (Sdh3). The membrane-integral protein Sdh3 plays specific functions in electron transfer in complex II. We show by genetic and biochemical approaches that Sdh3 also plays specific functions in the TIM22 complex. Sdh3 forms a subcomplex with Tim18 and is involved in biogenesis and assembly of the membrane-integral subunits of the TIM22 complex. We conclude that the assembly of Sdh3 with different partner proteins, Sdh4 and Tim18, recruits it to two different mitochondrial membrane complexes with functions in bioenergetics and protein biogenesis, respectively.
Subject(s)
Electron Transport , Mitochondrial Membrane Transport Proteins/metabolism , Mitochondrial Membranes/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Succinate Dehydrogenase/metabolism , Electron Transport Complex II/metabolism , Mitochondrial Membranes/enzymology , Mitochondrial Precursor Protein Import Complex Proteins , Saccharomyces cerevisiae/cytology , Saccharomyces cerevisiae/enzymologyABSTRACT
Mitochondrial Hsp70 (mtHsp70) mediates essential functions for mitochondrial biogenesis, like import and folding of proteins. In these processes, the chaperone cooperates with cochaperones, the presequence translocase, and other chaperone systems. The chaperonin Hsp60, together with its cofactor Hsp10, catalyzes folding of a subset of mtHsp70 client proteins. Hsp60 forms heptameric ring structures that provide a cavity for protein folding. How the Hsp60 rings are assembled is poorly understood. In a comprehensive interaction study, we found that mtHsp70 associates with Hsp60 and Hsp10. Surprisingly, mtHsp70 interacts with Hsp10 independently of Hsp60. The mtHsp70-Hsp10 complex binds to the unassembled Hsp60 precursor to promote its assembly into mature Hsp60 complexes. We conclude that coupling to Hsp10 recruits mtHsp70 to mediate the biogenesis of the heptameric Hsp60 rings.
Subject(s)
Chaperonin 10/metabolism , Chaperonin 60/metabolism , HSP70 Heat-Shock Proteins/metabolism , Mitochondria/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Animals , Chaperonin 60/chemistry , Saccharomyces cerevisiae/cytologyABSTRACT
The mitochondrial outer membrane contains integral α-helical and ß-barrel proteins that are imported from the cytosol. The machineries importing ß-barrel proteins have been identified, however, different views exist on the import of α-helical proteins. It has been reported that the biogenesis of Om45, the most abundant signal-anchored protein, does not depend on proteinaceous components, but involves direct insertion into the outer membrane. We show that import of Om45 occurs via the translocase of the outer membrane and the presequence translocase of the inner membrane. Assembly of Om45 in the outer membrane involves the MIM machinery. Om45 thus follows a new mitochondrial biogenesis pathway that uses elements of the presequence import pathway to direct a protein to the outer membrane.
Subject(s)
Carrier Proteins/metabolism , Membrane Proteins/metabolism , Mitochondrial Membrane Transport Proteins/metabolism , Mitochondrial Membranes/physiology , Protein Transport/physiology , Saccharomyces cerevisiae Proteins/metabolism , Autoradiography , Electrophoresis, Polyacrylamide Gel , Membrane Proteins/chemistry , Membrane Transport Proteins/metabolism , Mitochondrial Precursor Protein Import Complex Proteins , Mutagenesis , Polymerase Chain Reaction , Protein Structure, Secondary , Saccharomyces cerevisiae , Saccharomyces cerevisiae Proteins/chemistryABSTRACT
The conserved MIA pathway is responsible for the import and oxidative folding of proteins destined for the intermembrane space of mitochondria. In contrast to a wealth of information obtained from studies with yeast, the function of the MIA pathway in higher eukaryotes has remained enigmatic. Here, we took advantage of the molecular understanding of the MIA pathway in yeast and designed a model of the human MIA pathway. The yeast model for MIA consists of two critical components, the disulfide bond carrier Mia40 and sulfhydryl oxidase Erv1/ALR. Human MIA40 and ALR substituted for their yeast counterparts in the essential function for the oxidative biogenesis of mitochondrial intermembrane space proteins. In addition, the sulfhydryl oxidases ALR/Erv1 were found to be involved in the mitochondrial localization of human MIA40. Furthermore, the defective accumulation of human MIA40 in mitochondria underlies a recently identified disease that is caused by amino acid exchange in ALR. Thus, human ALR is an important factor that controls not only the ability of MIA40 to bind and oxidize protein clients but also the localization of human MIA40 in mitochondria.
Subject(s)
Mitochondrial Membrane Transport Proteins/metabolism , Mitochondrial Proteins/metabolism , Oxidoreductases Acting on Sulfur Group Donors/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Disulfides , Humans , Mitochondria/metabolism , Mitochondrial Membrane Transport Proteins/chemistry , Mitochondrial Membrane Transport Proteins/genetics , Mitochondrial Membranes/metabolism , Mitochondrial Precursor Protein Import Complex Proteins , Mitochondrial Proteins/genetics , Oxidoreductases Acting on Sulfur Group Donors/genetics , Protein Transport , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/geneticsABSTRACT
The majority of mitochondrial proteins are synthesized with amino-terminal signal sequences. The presequence translocase of the inner membrane (TIM23 complex) mediates the import of these preproteins. The essential TIM23 core complex closely cooperates with partner protein complexes like the presequence translocase-associated import motor and the respiratory chain. The inner mitochondrial membrane also contains a large number of metabolite carriers, but their association with preprotein translocases has been controversial. We performed a comprehensive analysis of the TIM23 interactome based on stable isotope labeling with amino acids in cell culture. Subsequent biochemical studies on identified partner proteins showed that the mitochondrial ADP/ATP carrier associates with the membrane-embedded core of the TIM23 complex in a stoichiometric manner, revealing an unexpected connection of mitochondrial protein biogenesis to metabolite transport. Our data indicate that direct TIM23-AAC coupling may support preprotein import into mitochondria when respiratory activity is low.
Subject(s)
Membrane Transport Proteins/metabolism , Mitochondrial Membranes/metabolism , Mitochondrial Proteins/metabolism , Multiprotein Complexes/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Membrane Transport Proteins/genetics , Mitochondrial Precursor Protein Import Complex Proteins , Mitochondrial Proteins/genetics , Multiprotein Complexes/genetics , Protein Transport/physiology , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/geneticsABSTRACT
The yeast protein Zim17 belongs to a unique class of co-chaperones that maintain the solubility of Hsp70 proteins in mitochondria and plastids of eukaryotic cells. However, little is known about the functional cooperation between Zim17 and mitochondrial Hsp70 proteins in vivo. To analyze the effects of a loss of Zim17 function in the authentic environment, we introduced novel conditional mutations within the ZIM17 gene of the model organism Saccharomyces cerevisiae that allowed a recovery of temperature-sensitive but respiratory competent zim17 mutant cells. On fermentable growth medium, the mutant cells were prone to acquire respiratory deficits and showed a strong aggregation of the mitochondrial Hsp70 Ssq1 together with a concomitant defect in Fe/S protein biogenesis. In contrast, under respiring conditions, the mitochondrial Hsp70s Ssc1 and Ssq1 exhibited only a partial aggregation. We show that the induction of the zim17 mutant phenotype leads to strong import defects for Ssc1-dependent matrix-targeted precursor proteins that correlate with a significantly reduced binding of newly imported substrate proteins to Ssc1. We conclude that Zim17 is not only required for the maintenance of mtHsp70 solubility but also directly assists the functional interaction of mtHsp70 with substrate proteins in a J-type co-chaperone-dependent manner.
Subject(s)
HSP70 Heat-Shock Proteins/metabolism , Iron/metabolism , Mitochondrial Membranes/metabolism , Mitochondrial Proteins/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Sulfur/metabolism , HSP70 Heat-Shock Proteins/genetics , Mitochondrial Proteins/genetics , Mutation , Protein Binding/physiology , Protein Precursors/genetics , Protein Precursors/metabolism , Protein Transport/physiology , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/geneticsABSTRACT
The mitochondrial inner membrane harbors the complexes of the respiratory chain and protein translocases required for the import of mitochondrial precursor proteins. These complexes are functionally interdependent, as the import of respiratory chain precursor proteins across and into the inner membrane requires the membrane potential. Vice versa the membrane potential is generated by the proton pumping complexes of the respiratory chain. Besides this basic codependency four different systems for protein import, processing and assembly show further connections to the respiratory chain. The mitochondrial intermembrane space import and assembly machinery oxidizes cysteine residues within the imported precursor proteins and is able to donate the liberated electrons to the respiratory chain. The presequence translocase of the inner membrane physically interacts with the respiratory chain. The mitochondrial processing peptidase is homologous to respiratory chain subunits and the carrier translocase of the inner membrane even shares a subunit with the respiratory chain. In this review we will summarize the import of mitochondrial precursor proteins and highlight these special links between the mitochondrial protein import machinery and the respiratory chain. This article is part of a Special Issue entitled: Respiratory complex II: Role in cellular physiology and disease.
Subject(s)
Membrane Potential, Mitochondrial/physiology , Mitochondrial Membrane Transport Proteins/metabolism , Mitochondrial Membranes/metabolism , Mitochondrial Proteins/metabolism , Electron Transport/physiology , Electron Transport Complex II/metabolism , Humans , Models, Biological , Protein Transport/physiologyABSTRACT
The mitochondrial inner membrane is the central energy-converting membrane of eukaryotic cells. The electrochemical proton gradient generated by the respiratory chain drives the ATP synthase. To maintain this proton-motive force, the inner membrane forms a tight barrier and strictly controls the translocation of ions. However, the major preprotein transport machinery of the inner membrane, termed the presequence translocase, translocates polypeptide chains into or across the membrane. Different views exist of the molecular mechanism of the translocase, in particular of the coupling with the import motor of the matrix. We have reconstituted preprotein transport into the mitochondrial inner membrane by incorporating the purified presequence translocase into cardiolipin-containing liposomes. We show that the motor-free form of the presequence translocase integrates preproteins into the membrane. The reconstituted presequence translocase responds to targeting peptides and mediates voltage-driven preprotein translocation, lateral release and insertion into the lipid phase. Thus, the minimal system for preprotein integration into the mitochondrial inner membrane is the presequence translocase, a cardiolipin-rich membrane and a membrane potential.
Subject(s)
Membrane Transport Proteins/metabolism , Mitochondrial Membrane Transport Proteins/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Cytochromes c1/metabolism , Immunoprecipitation , Membrane Potential, Mitochondrial/physiology , Membrane Transport Proteins/genetics , Mitochondria/metabolism , Mitochondrial Membrane Transport Proteins/genetics , Mitochondrial Membranes/metabolism , Mitochondrial Precursor Protein Import Complex Proteins , Protein Precursors/metabolism , Protein Transport , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/geneticsABSTRACT
Cytochrome c assembly requires sulphydryls at the CXXCH haem binding site on the apoprotein and also chemical reduction of the haem co-factor. In yeast mitochondria, the cytochrome haem lyases (CCHL, CC(1) HL) and Cyc2p catalyse covalent haem attachment to apocytochromes c and c(1) . An in vivo indication that Cyc2p controls a reductive step in the haem attachment reaction is the finding that the requirement for its function can be bypassed by exogenous reductants. Although redox titrations of Cyc2p flavin (E(m) = -290 mV) indicate that reduction of a disulphide at the CXXCH site of apocytochrome c (E(m) = -265 mV) is a thermodynamically favourable reaction, Cyc2p does not act as an apocytochrome c or c(1) CXXCH disulphide reductase in vitro. In contrast, Cyc2p is able to catalyse the NAD(P)H-dependent reduction of hemin, an indication that the protein's role may be to control the redox state of the iron in the haem attachment reaction to apocytochromes c. Using two-hybrid analysis, we show that Cyc2p interacts with CCHL and also with apocytochromes c and c(1) . We postulate that Cyc2p, possibly in a complex with CCHL, reduces the haem iron prior to haem attachment to the apoforms of cytochrome c and c(1) .
Subject(s)
Carrier Proteins/metabolism , Mitochondrial Proteins/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/enzymology , Cytochromes c/metabolism , Lyases/metabolism , NADP/metabolism , Two-Hybrid System TechniquesABSTRACT
The presequence translocase (TIM23 complex) imports precursor proteins into the mitochondrial inner membrane and matrix. The presequence translocase-associated motor (PAM) provides a driving force for transport into the matrix. The J-protein Pam18 stimulates the ATPase activity of the mitochondrial Hsp70 (mtHsp70). Pam16 recruits Pam18 to the TIM23 complex to ensure protein import. The Pam16-Pam18 module also associates with components of the respiratory chain, but the function of the dual localization of Pam16-Pam18 is largely unknown. Here, we show that disruption of the Pam16-Pam18 heterodimer causes redistribution of Pam18 to the respiratory chain supercomplexes, where it forms a homodimer. Redistribution of Pam18 decreases protein import into mitochondria but stimulates mtHsp70-dependent assembly of respiratory chain complexes. We conclude that coupling to Pam16 differentially controls the dual function of Pam18. It recruits Pam18 to the TIM23 complex to promote protein import but attenuates the Pam18 function in the assembly of respiratory chain complexes.
Subject(s)
Membrane Transport Proteins , Mitochondrial Precursor Protein Import Complex Proteins , Saccharomyces cerevisiae Proteins , Carrier Proteins/metabolism , Electron Transport , HSP70 Heat-Shock Proteins/metabolism , Membrane Transport Proteins/metabolism , Mitochondrial Membrane Transport Proteins/metabolism , Mitochondrial Precursor Protein Import Complex Proteins/metabolism , Protein Transport , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/metabolismABSTRACT
Saccharomyces cerevisiae Mdm38 and Ylh47 are homologues of human Letm1, a protein implicated in Wolf-Hirschhorn syndrome. We analyzed the function of Mdm38 and Ylh47 in yeast mitochondria to gain insight into the role of Letm1. We find that mdm38Delta mitochondria have reduced amounts of certain mitochondrially encoded proteins and low levels of complex III and IV and accumulate unassembled Atp6 of complex V of the respiratory chain. Mdm38 is especially required for efficient transport of Atp6 and cytochrome b across the inner membrane, whereas Ylh47 plays a minor role in this process. Both Mdm38 and Ylh47 form stable complexes with mitochondrial ribosomes, similar to what has been reported for Oxa1, a central component of the mitochondrial export machinery. Our results indicate that Mdm38 functions as a component of an Oxa1-independent insertion machinery in the inner membrane and that Mdm38 plays a critical role in the biogenesis of the respiratory chain by coupling ribosome function to protein transport across the inner membrane.
Subject(s)
Membrane Proteins/metabolism , Mitochondria/metabolism , Mitochondrial Membranes/metabolism , Mitochondrial Proteins/metabolism , Ribosomes/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Calcium-Binding Proteins/metabolism , Cytochromes b/metabolism , Electron Transport Complex IV/metabolism , Humans , Membrane Proteins/genetics , Mitochondrial Proton-Translocating ATPases/metabolism , Nuclear Proteins/metabolism , Protein Transport/physiology , Saccharomyces cerevisiae Proteins/genetics , Signal TransductionABSTRACT
The mitochondrial intermembrane space contains a family of small Tim proteins that function as essential chaperones for protein import. The soluble Tim9-Tim10 complex transfers hydrophobic precursor proteins through the aqueous intermembrane space to the carrier translocase of the inner membrane (TIM22 complex). Tim12, a peripheral membrane subunit of the TIM22 complex, is thought to recruit a portion of Tim9-Tim10 to the inner membrane. It is not known, however, how Tim12 is assembled. We have identified a new intermediate in the biogenesis pathway of Tim12. A soluble form of Tim12 first assembles with Tim9 and Tim10 to form a Tim12-core complex. Tim12-core then docks onto the membrane-integrated subunits of the TIM22 complex to form the holo-translocase. Thus, the function of Tim12 in linking soluble and membrane-integrated subunits of the import machinery involves a sequential assembly mechanism of the translocase through a soluble intermediate complex of the three essential small Tim proteins.
Subject(s)
Membrane Proteins/metabolism , Membrane Transport Proteins/metabolism , Mitochondrial ADP, ATP Translocases/metabolism , Mitochondrial Proteins/metabolism , Protein Subunits/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Membrane Proteins/genetics , Membrane Transport Proteins/genetics , Mitochondrial Membrane Transport Proteins , Mitochondrial Precursor Protein Import Complex Proteins , Mitochondrial Proteins/genetics , Multiprotein Complexes/metabolism , Protein Precursors/genetics , Protein Precursors/metabolism , Protein Subunits/genetics , Saccharomyces cerevisiae/cytology , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/geneticsABSTRACT
The import of mitochondrial preproteins requires an electric potential across the inner membrane and the hydrolysis of ATP in the matrix. We assessed the contributions of the two energy sources to the translocation driving force responsible for movement of the polypeptide chain through the translocation channel and the unfolding of preprotein domains. The import-driving activity was directly analyzed by the determination of the protease resistances of saturating amounts of membrane-spanning translocation intermediates. The ability to generate a strong translocation-driving force was solely dependent on the activity of the ATP-dependent import motor complex in the matrix. For a sustained import-driving activity on the preprotein in transit, an unstructured N-terminal segment of more than 70 to 80 amino acid residues was required. The electric potential of the inner membrane was required to maintain the import-driving activity at a high level. The electrophoretic force of the potential exhibited only a limited capacity to unfold preprotein domains. We conclude that the membrane potential increases the probability of a dynamic interaction of the preprotein with the import motor. Polypeptide translocation and unfolding are mainly driven by the inward-directed translocation activity based on the functional cooperation of the import motor components.
Subject(s)
Adenosine Triphosphate/metabolism , Membrane Potential, Mitochondrial/physiology , Mitochondrial Membrane Transport Proteins/physiology , Molecular Motor Proteins/physiology , Protein Precursors/metabolism , Carrier Proteins/genetics , Carrier Proteins/metabolism , L-Lactate Dehydrogenase (Cytochrome)/genetics , L-Lactate Dehydrogenase (Cytochrome)/metabolism , Membrane Transport Proteins/genetics , Membrane Transport Proteins/metabolism , Mitochondrial Membrane Transport Proteins/genetics , Mitochondrial Precursor Protein Import Complex Proteins , Molecular Motor Proteins/genetics , Mutation , Peptides/genetics , Peptides/metabolism , Protein Folding , Protein Precursors/genetics , Protein Structure, Tertiary , Protein Transport , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Saccharomyces cerevisiae , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Submitochondrial Particles/genetics , Submitochondrial Particles/physiology , Tetrahydrofolate Dehydrogenase/genetics , Tetrahydrofolate Dehydrogenase/metabolismABSTRACT
Mitochondrial preproteins contain amino-terminal presequences directing them to the presequence translocase of the mitochondrial inner membrane (TIM23 complex). Depending on additional downstream import signals, TIM23 either inserts preproteins into the inner membrane or translocates them into the matrix. Matrix import requires the coupling of the presequence translocase-associated motor (PAM) to TIM23. The molecular mechanisms coordinating preprotein recognition by TIM23 in the intermembrane space (IMS) with PAM activation in the matrix are unknown. Here we show that subsequent to presequence recognition in the IMS, the Tim50 matrix domain facilitates the recruitment of the coupling factor Pam17. Next, the IMS domain of Tim50 promotes PAM recruitment to TIM23. Finally, the Tim50 transmembrane segment stimulates the matrix-directed import-driving force exerted by PAM. We propose that recognition of preprotein segments in the IMS and transfer of signal information across the inner membrane by Tim50 determine import motor activation.
Subject(s)
Cell Membrane/metabolism , Mitochondria/metabolism , Mitochondrial Membrane Transport Proteins/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Mitochondrial Membrane Transport Proteins/chemistry , Mitochondrial Precursor Protein Import Complex Proteins , Models, Biological , Molecular Motor Proteins/metabolism , Protein Domains , Protein Transport , Saccharomyces cerevisiae Proteins/chemistryABSTRACT
In mitochondria, the carrier translocase (TIM22 complex) facilitates membrane insertion of multi-spanning proteins with internal targeting signals into the inner membrane [1-3]. Tom70, a subunit of TOM complex, represents the major receptor for these precursors [2, 4-6]. After transport across the outer membrane, the hydrophobic carriers engage with the small TIM protein complex composed of Tim9 and Tim10 for transport across the intermembrane space (IMS) toward the TIM22 complex [7-12]. Tim22 represents the pore-forming core unit of the complex [13, 14]. Only a small subset of TIM22 cargo molecules, containing four or six transmembrane spans, have been experimentally defined. Here, we used a tim22 temperature-conditional mutant to define the TIM22 substrate spectrum. Along with carrier-like cargo proteins, we identified subunits of the mitochondrial pyruvate carrier (MPC) as unconventional TIM22 cargos. MPC proteins represent substrates with atypical topology for this transport pathway. In agreement with this, a patient affected in TIM22 function displays reduced MPC levels. Our findings broaden the repertoire of carrier pathway substrates and challenge current concepts of TIM22-mediated transport processes.
Subject(s)
Membrane Transport Proteins/genetics , Mitochondrial Proteins/genetics , Monocarboxylic Acid Transporters/genetics , Pyruvic Acid/metabolism , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae/physiology , Biological Transport , HEK293 Cells , Humans , Membrane Transport Proteins/metabolism , Mitochondrial Precursor Protein Import Complex Proteins , Mitochondrial Proteins/metabolism , Monocarboxylic Acid Transporters/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/metabolismABSTRACT
The mitochondrial outer membrane contains integral proteins with α-helical membrane anchors or a transmembrane ß-barrel. The translocase of the outer membrane (TOM) cooperates with the sorting and assembly machinery (SAM) in the import of ß-barrel proteins, whereas the mitochondrial import (MIM) complex inserts precursors of multi-spanning α-helical proteins. Single-spanning proteins constitute more than half of the integral outer membrane proteins; however, their biogenesis is poorly understood. We report that the yeast MIM complex promotes the insertion of proteins with N-terminal (signal-anchored) or C-terminal (tail-anchored) membrane anchors. The MIM complex exists in three dynamic populations. MIM interacts with TOM to accept precursor proteins from the receptor Tom70. Free MIM complexes insert single-spanning proteins that are imported in a Tom70-independent manner. Finally, coupling of MIM and SAM promotes early assembly steps of TOM subunits. We conclude that the MIM complex is a major and versatile protein translocase of the mitochondrial outer membrane.