ABSTRACT
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
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.
Subject(s)
Membrane Proteins/metabolism , Mitochondrial Membrane Transport Proteins/metabolism , Mitochondrial Membranes/metabolism , Humans , Protein Conformation, alpha-HelicalABSTRACT
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
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
The presequence translocase of the mitochondrial inner membrane (TIM23 complex) facilitates anterograde precursor transport into the matrix and lateral release of precursors with stop-transfer signal into the membrane (sorting). Sorting requires precursor exit from the translocation channel into the lipid phase through the lateral gate of the TIM23 complex. How the two transport modes are regulated and balanced against each other is unknown. Here we show that the import motor J-protein Pam18, which is essential for matrix import, controls lateral protein release into the lipid bilayer. Constitutively translocase-associated Pam18 obstructs lateral precursor transport. Concomitantly, Mgr2, implicated in precursor quality control, is displaced from the translocase. We conclude that during motor-dependent matrix protein transport, the transmembrane segment of Pam18 closes the lateral gate to promote anterograde polypeptide movement. This finding explains why a motor-free form of the translocase facilitates the lateral movement of precursors with a stop-transfer signal.
Subject(s)
Membrane Transport Proteins/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Mitochondrial Membranes/metabolism , Mitochondrial Precursor Protein Import Complex Proteins , YeastsABSTRACT
The biogenesis of mitochondria, chloroplasts, and Gram-negative bacteria requires the insertion of ß-barrel proteins into the outer membranes. Homologous Omp85 proteins are essential for membrane insertion of ß-barrel precursors. It is unknown if precursors are threaded through the Omp85-channel interior and exit laterally or if they are translocated into the membrane at the Omp85-lipid interface. We have mapped the interaction of a precursor in transit with the mitochondrial Omp85-channel Sam50 in the native membrane environment. The precursor is translocated into the channel interior, interacts with an internal loop, and inserts into the lateral gate by ß-signal exchange. Transport through the Omp85-channel interior followed by release through the lateral gate into the lipid phase may represent a basic mechanism for membrane insertion of ß-barrel proteins.
Subject(s)
Mitochondria/metabolism , Mitochondrial Membrane Transport Proteins/metabolism , Mitochondrial Membranes/metabolism , Mitochondrial Proteins/metabolism , Porins/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Voltage-Dependent Anion Channel 1/metabolism , Mitochondrial Membrane Transport Proteins/genetics , Mitochondrial Proteins/chemistry , Mitochondrial Proteins/genetics , Porins/genetics , Protein Conformation, beta-Strand , Protein Folding , Protein Transport , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/genetics , Voltage-Dependent Anion Channel 1/geneticsABSTRACT
Two driving forces energize precursor translocation across the inner mitochondrial membrane. Although the membrane potential (Δψ) is considered to drive translocation of positively charged presequences through the TIM23 complex (presequence translocase), the activity of the Hsp70-powered import motor is crucial for the translocation of the mature protein portion into the matrix. In this study, we show that mitochondrial matrix proteins display surprisingly different dependencies on the Δψ. However, a precursor's hypersensitivity to a reduction of the Δψ is not linked to the respective presequence, but rather to the mature portion of the polypeptide chain. The presequence translocase constituent Pam17 is specifically recruited by the receptor Tim50 to promote the transport of hypersensitive precursors into the matrix. Our analyses show that two distinct Δψ-driven translocation steps energize precursor passage across the inner mitochondrial membrane. The Δψ- and Pam17-dependent import step identified in this study is positioned between the two known energy-dependent steps: Δψ-driven presequence translocation and adenosine triphosphate-driven import motor activity.
Subject(s)
Membrane Potential, Mitochondrial , Membrane Proteins/metabolism , Mitochondria/metabolism , Mitochondrial Membrane Transport Proteins/metabolism , Mitochondrial Membranes/metabolism , Protein Precursors/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Adenosine Triphosphate/metabolism , Genotype , Hydrolysis , Membrane Proteins/genetics , Mitochondrial Membrane Transport Proteins/genetics , Mitochondrial Precursor Protein Import Complex Proteins , Mutation , Phenotype , Protein Precursors/genetics , Protein Transport , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/genetics , Time FactorsABSTRACT
The endoplasmic reticulum-mitochondria encounter structure (ERMES) connects the mitochondrial outer membrane with the ER. Multiple functions have been linked to ERMES, including maintenance of mitochondrial morphology, protein assembly and phospholipid homeostasis. Since the mitochondrial distribution and morphology protein Mdm10 is present in both ERMES and the mitochondrial sorting and assembly machinery (SAM), it is unknown how the ERMES functions are connected on a molecular level. Here we report that conserved surface areas on opposite sides of the Mdm10 ß-barrel interact with SAM and ERMES, respectively. We generated point mutants to separate protein assembly (SAM) from morphology and phospholipid homeostasis (ERMES). Our study reveals that the ß-barrel channel of Mdm10 serves different functions. Mdm10 promotes the biogenesis of α-helical and ß-barrel proteins at SAM and functions as integral membrane anchor of ERMES, demonstrating that SAM-mediated protein assembly is distinct from ER-mitochondria contact sites.
Subject(s)
Endoplasmic Reticulum/metabolism , Membrane Proteins/metabolism , Mitochondrial Proteins/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Membrane Proteins/chemistry , Mitochondria/metabolism , Mitochondrial Membrane Transport Proteins/metabolism , Mitochondrial Membranes/metabolism , Mitochondrial Proteins/chemistry , Models, Biological , Protein Binding , Protein Interaction Domains and Motifs , Protein Structure, Secondary , Saccharomyces cerevisiae Proteins/chemistryABSTRACT
The highly organized mitochondrial inner membrane harbors enzymes that produce the bulk of cellular ATP via oxidative phosphorylation. The majority of inner membrane protein precursors are synthesized in the cytosol. Precursors with a cleavable presequence are imported by the presequence translocase (TIM23 complex), while other precursors containing internal targeting signals are imported by the carrier translocase (TIM22 complex). Both TIM23 and TIM22 are activated by the transmembrane electrochemical potential. Many small inner membrane proteins, however, do not resemble canonical TIM23 or TIM22 substrates and their mechanism of import is unknown. We report that subunit e of the F1Fo-ATP synthase, a small single-spanning inner membrane protein that is critical for inner membrane organization, is imported by TIM23 in a process that does not require activation by the membrane potential. Absence of positively charged residues at the matrix-facing amino-terminus of subunit e facilitates membrane potential-independent import. Instead, engineered positive charges establish a dependence of the import reaction on the electrochemical potential. Our results have two major implications. First, they reveal an unprecedented pathway of protein import into the mitochondrial inner membrane, which is mediated by TIM23. Second, they directly demonstrate the role of the membrane potential in driving the electrophoretic transport of positively charged protein segments across the inner membrane.
Subject(s)
Membrane Potentials , Membrane Transport Proteins/metabolism , Mitochondria/metabolism , Mitochondria/physiology , Mitochondrial Proton-Translocating ATPases/metabolism , Saccharomyces cerevisiae Proteins/metabolism , DNA Mutational Analysis , Mitochondrial Precursor Protein Import Complex Proteins , Mitochondrial Proton-Translocating ATPases/genetics , Protein Transport , Saccharomyces cerevisiae Proteins/geneticsABSTRACT
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 transcriptional regulator HAP4, induced by respiratory substrates, is involved in the balance between fermentation and respiration in S. cerevisiae. We identified putative orthologues of the Hap4 protein in all ascomycetes, based only on a conserved sixteen amino acid-long motif. In addition to this motif, some of these proteins contain a DNA-binding motif of the bZIP type, while being nonetheless globally highly divergent. The genome of the yeast Hansenula polymorpha contains two HAP4-like genes encoding the protein HpHap4-A which, like ScHap4, is devoid of a bZIP motif, and HpHap4-B which contains it. This species has been chosen for a detailed examination of their respective properties. Based mostly on global gene expression studies performed in the S. cerevisiae HAP4 disruption mutant (ScΔhap4), we show here that HpHap4-A is functionally equivalent to ScHap4, whereas HpHap4-B is not. Moreover HpHAP4-B is able to complement the H2O2 hypersensitivity of the ScYap1 deletant, YAP1 being, in S. cerevisiae, the main regulator of oxidative stress. Finally, a transcriptomic analysis performed in the ScΔyap1 strain overexpressing HpHAP4-B shows that HpHap4-B acts both on oxidative stress response and carbohydrate metabolism in a manner different from both ScYap1 and ScHap4. Deletion of these two genes in their natural host, H. polymorpha, confirms that HpHAP4-A participates in the control of the fermentation/respiration balance, while HpHAP4-B is involved in oxidative stress since its deletion leads to hypersensitivity to H2O2. These data, placed in an evolutionary context, raise new questions concerning the evolution of the HAP4 transcriptional regulation function and suggest that Yap1 and Hap4 have diverged from a unique regulatory protein in the fungal ancestor.
Subject(s)
CCAAT-Binding Factor/genetics , Oxidative Stress/genetics , Saccharomyces cerevisiae Proteins/genetics , Transcription Factors/genetics , Transcription, Genetic , Amino Acid Motifs/genetics , CCAAT-Binding Factor/metabolism , Carbon/metabolism , Gene Expression Regulation, Fungal , Genome, Fungal , Hydrogen Peroxide/chemistry , Oxidation-Reduction , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/metabolism , Transcription Factors/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
Mitochondria play central roles in cellular energy conversion, metabolism, and apoptosis. Mitochondria import more than 1000 different proteins from the cytosol. It is unknown if the mitochondrial protein import machinery is connected to the cell division cycle. We found that the cyclin-dependent kinase Cdk1 stimulated assembly of the main mitochondrial entry gate, the translocase of the outer membrane (TOM), in mitosis. The molecular mechanism involved phosphorylation of the cytosolic precursor of Tom6 by cyclin Clb3-activated Cdk1, leading to enhanced import of Tom6 into mitochondria. Tom6 phosphorylation promoted assembly of the protein import channel Tom40 and import of fusion proteins, thus stimulating the respiratory activity of mitochondria in mitosis. Tom6 phosphorylation provides a direct means for regulating mitochondrial biogenesis and activity in a cell cycle-specific manner.
Subject(s)
Cell Cycle , Mitochondria/metabolism , Mitochondrial Membrane Transport Proteins/metabolism , Protein Precursors/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/cytology , Saccharomyces cerevisiae/metabolism , CDC2 Protein Kinase/metabolism , Cyclin B/metabolism , Cytosol/metabolism , Mitochondrial Precursor Protein Import Complex Proteins , Phosphorylation , Protein TransportABSTRACT
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 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
Most mitochondrial proteins are imported by the translocase of the outer mitochondrial membrane (TOM). Tom22 functions as central receptor and transfers preproteins to the import pore. Casein kinase 2 (CK2) constitutively phosphorylates the cytosolic precursor of Tom22 at Ser44 and Ser46 and, thus, promotes its import. It is unknown whether Tom22 is regulated under different metabolic conditions. We report that CK1, which is involved in glucose-induced signal transduction, is bound to mitochondria. CK1 phosphorylates Tom22 at Thr57 and stimulates the assembly of Tom22 and Tom20. In contrast, protein kinase A (PKA), which is also activated by the addition of glucose, phosphorylates the precursor of Tom22 at Thr76 and impairs its import. Thus, PKA functions in an opposite manner to CK1 and CK2. Our results reveal that three kinases regulate the import and assembly of Tom22, demonstrating that the central receptor is a major target for the posttranslational regulation of mitochondrial protein import.
Subject(s)
Glucose/pharmacology , Mitochondria/drug effects , Mitochondrial Membrane Transport Proteins/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Casein Kinase I/metabolism , Casein Kinase II/metabolism , Cyclic AMP-Dependent Protein Kinases/metabolism , Mitochondria/enzymology , Mitochondria/metabolism , Mitochondrial Membranes/metabolism , Phosphorylation/drug effects , Protein Binding , Saccharomyces cerevisiae/metabolism , Signal Transduction/drug effectsABSTRACT
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 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
The formation of the mature cytochrome c oxidase (complex IV) involves the association of nuclear- and mitochondria-encoded subunits. The assembly of nuclear-encoded subunits like cytochrome c oxidase subunit 4 (Cox4) into the mature complex is poorly understood. Cox4 is crucial for the stability of complex IV. To find specific biogenesis factors, we analyze interaction partners of Cox4 by affinity purification and mass spectroscopy. Surprisingly, we identify a complex of Cox4, the mitochondrial Hsp70 (mtHsp70), and its nucleotide-exchange factor mitochondrial GrpE (Mge1). We generate a yeast mutant of mtHsp70 specifically impaired in the formation of this novel mtHsp70-Mge1-Cox4 complex. Strikingly, the assembly of Cox4 is strongly decreased in these mutant mitochondria. Because Cox4 is a key factor for the biogenesis of complex IV, we conclude that the mtHsp70-Mge1-Cox4 complex plays an important role in the formation of cytochrome c oxidase. Cox4 arrests at this chaperone complex in the absence of mature complex IV. Thus the mtHsp70-Cox4 complex likely serves as a novel delivery system to channel Cox4 into the assembly line when needed.
Subject(s)
Electron Transport Complex IV/metabolism , HSP70 Heat-Shock Proteins/metabolism , Mitochondrial Membrane Transport Proteins/metabolism , Molecular Chaperones/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Carrier Proteins/chemistry , Carrier Proteins/metabolism , Electron Transport Complex IV/chemistry , HSP70 Heat-Shock Proteins/genetics , Membrane Proteins/metabolism , Mitochondria/metabolism , Mitochondrial Membrane Transport Proteins/chemistry , Mitochondrial Proteins/chemistry , Mitochondrial Proteins/metabolism , Molecular Chaperones/chemistry , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/chemistry , 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.