The protein chaperone Ssa1 affects mRNA localization to the mitochondria

Many nuclear-transcribed mRNAs encoding mitochondrial proteins are localized near the mitochondrial outer membrane. A yet unresolved question is whether protein synthesis is important for transport of these mRNAs to their destination. Herein we present a connection between mRNA localization in yeast and the protein chaperone Ssa1. Ssa1 depletion lowered mRNA association with mitochondria while its overexpression increased it. A genome-wide analysis revealed that Ssa proteins preferentially affect mRNAs encoding hydrophobic proteins, which are expected targets for these protein chaperones. Importantly, deletion of the mitochondrial receptor Tom70 abolished the impact of Ssa1 overexpression on mRNAs encoding Tom70 targets. Taken together, our results suggest a role for Ssa1 in mediating localization of nascent peptide-ribosome-mRNA complexes to the mitochondria, consistent with a co-translational transport process.     

Localization of mRNAs coding for mitochondrial proteins in the yeast Saccharomyces cerevisiae

Targeted mRNA localization is a likely determinant of localized protein synthesis. To investigate whether mRNAs encoding mitochondrial proteins (mMPs) localize to mitochondria and, thus, might confer localized protein synthesis and import, we visualized endogenously expressed mMPs in vivo for the first time. We determined the localization of 24 yeast mMPs encoding proteins of the mitochondrial matrix, outer and inner membrane, and intermembrane space and found that many mMPs colocalize with mitochondria in vivo. This supports earlier cell fractionation and microarray-based studies that proposed mMP association with the mitochondrial fraction. Interestingly, a number of mMPs showed a dependency on the mitochondrial Puf3 RNA-binding protein, as well as nonessential proteins of the translocase of the outer membrane (TOM) complex import machinery, for normal colocalization with mitochondria. We examined the specific determinants of ATP2 and OXA1 mRNA localization and found a mutual dependency on the 3' UTR, Puf3, Tom7, and Tom70, but not Tom20, for localization. Tom6 may facilitate the localization of specific mRNAs as OXA1, but not ATP2, mRNA was mislocalized in tom6Δ cells. Interestingly, a substantial fraction of OXA1 and ATP2 RNA granules colocalized with the endoplasmic reticulum (ER) and a deletion in MDM10, which mediates mitochondria-ER tethering, resulted in a significant loss of OXA1 mRNA localization with ER. Finally, neither ATP2 nor OXA1 mRNA targeting was affected by a block in translation initiation, indicating that translation may not be essential for mRNA anchoring. Thus, endogenously expressed mRNAs are targeted to the mitochondria in vivo, and multiple factors contribute to mMP localization.     

Tom20 Mediates Localization of mRNAs to Mitochondria in a Translation-Dependent Manner

mRNAs encoding mitochondrial proteins are enriched in the vicinity of mitochondria, presumably to facilitate protein transport. A possible mechanism for enrichment may involve interaction of the translocase of the mitochondrial outer membrane (TOM) complex with the precursor protein while it is translated, thereby leading to association of polysomal mRNAs with mitochondria. To test this hypothesis, we isolated mitochondrial fractions from yeast cells lacking the major import receptor, Tom20, and compared their mRNA repertoire to that of wild-type cells by DNA microarrays. Most mRNAs encoding mitochondrial proteins were less associated with mitochondria, yet the extent of decrease varied among genes. Analysis of several mRNAs revealed that optimal association of Tom20 target mRNAs requires both translating ribosomes and features within the encoded mitochondrial targeting signal. Recently, Puf3p was implicated in the association of mRNAs with mitochondria through interaction with untranslated regions. We therefore constructed a tom20Δ puf3Δ double-knockout strain, which demonstrated growth defects under conditions where fully functional mitochondria are required. Mislocalization effects for few tested mRNAs appeared stronger in the double knockout than in the tom20Δ strain. Taken together, our data reveal a large-scale mRNA association mode that involves interaction of Tom20p with the translated mitochondrial targeting sequence and may be assisted by Puf3p.mRNA localization to distinct cellular compartments is important for the efficiency and specificity of the translation process. Synthesis of proteins at their sites of action may decrease the likelihood of ectopic protein expression and facilitate assembly of large multiprotein complexes. Two general modes for mRNA localization are known. The first, which is common for endoplasmic reticulum (ER)-associated mRNAs, necessitates translation of a short region of the protein (the signal peptide). The signal is recognized by the signal recognition particle as it emerges from the ribosome exit tunnel, and the complex that includes the mRNA, ribosome, and signal recognition particle is targeted to the ER (18). As an outcome of this process, mRNAs that encode proteins destined for the ER and the secretory pathway are associated with this compartment (7). The second mode for mRNA localization occurs prior to translation and in many cases prevents initiation of protein synthesis. Sequences or structural elements of the mRNA are bound by RNA-binding proteins, and these interact with transport factors, which direct the mRNA to its destination (5, 35, 42). Genome-wide studies indicate that localization by either mode is a broad phenomenon that encompasses many mRNAs and various cellular destinations (6, 21, 32, 38). Interestingly, we along with others have recently shown that noncoding regions may also be involved in localization of ER-associated mRNAs (1, 26, 38), demonstrating that these two modes are not mutually exclusive.Most of the mitochondrial proteins are encoded in the nucleus and need to be imported into the organelle. Various in vitro and in vivo assays led to the widely accepted notion that import may occur posttranslationally, i.e., after the protein is fully synthesized in the cytosol (33). However, mounting evidence also supports a cotranslational import of proteins into the mitochondria. Specifically, polysomes were shown to be associated with the mitochondrial surface, and these translated a distinct set of proteins (12, 19, 20). Moreover, isolated mitochondria are associated with many different mRNAs that encode mitochondrial proteins (28, 46). Elements from both the coding region (the mitochondrial targeting signal [MTS]) and the 3′ untranslated region (UTR) were shown to be important for targeting of some of these mRNAs (4, 29). One model for localization suggests association of the nascent peptide chain (specifically, the N-terminal MTS) with receptors on the mitochondria, coupled to cotranslational insertion of the protein (24). As an outcome of this cotranslational mechanism, polysomal mRNAs become associated with the mitochondria, analogously to what is observed in the ER. However, experimental support for this hypothesis is currently lacking.Recently, Saint-Georges et al. (41) have shown a role for Puf3p in localization of many mRNAs to the mitochondria of Saccharomyces cerevisiae. Puf3p is associated with the mitochondria outer membrane (11), and its role is mediated through interaction with UTRs. This may suggest a translation-independent mode of action. Intriguingly, however, most Puf3 targets appeared to be mislocalized also after treatment with the translation inhibitor cycloheximide (CHX), suggesting that an active translation process is required for their asymmetric localization (41). Moreover, a large number of mRNAs that are not Puf3 targets appeared to be affected from treatments with the translation inhibitors puromycin and cycloheximide (41), further supporting the existence of an additional, translation-dependent mode of mRNA targeting to the mitochondria.The translocase of the mitochondrial outer membrane (TOM complex) is a multiprotein machinery which mediates the import of the vast majority of proteins into the mitochondria (36, 39). Its core protein (Tom40) forms a β-barrel structure and serves as the main component of the import pore. Tom20 is a peripheral component of the TOM complex that functions as a primary receptor for mitochondrial precursor proteins (15). It was hypothesized that protein receptors interact with the incoming polypeptide while it is translated, and this leads to a local increase in mRNA concentration (24). An open question is whether the TOM complex, through Tom20, interacts with polypeptides while they are translated and thereby leads to higher local concentrations of mRNAs near the mitochondria. To address this issue, we analyzed the effects of TOM20 deletion on mRNA association with mitochondrial fractions and the role of the MTS on mRNA localization. We also tested the interactions between Tom20 and Puf3. We found that Tom20 is involved in mitochondrial association of many mRNAs by a process that requires the MTS. Tom20 deletion affects the localization of Puf3p, and a strain with deletions of both Tom20 and Puf3 exhibits a growth defect under conditions that require mitochondrial optimal function.     

Complex formation and turnover of mitochondrial transporters and ion channels

Mitochondria are responsible for many vital cellular functions in eukaryotic cells, such as ATP production, steroid synthesis and prosthetic group biogenesis. The vital functions of mitochondria are possible due to the compartmental nature of this organelle. Mitochondria form a dynamic network that can exist as a network throughout a cell or as distinct individual structures. Mitochondria are also composed of two membranes, an inner and outer membrane. The inner mitochondrial membrane (IMM) is significantly larger than the outer membrane and must fold upon itself to be contained within the outer mitochondrial membrane (OMM). These folds are known as cristae. Altogether these different membrane compartments specialize in different functions of the mitochondria. The OMM is responsible for passage of small metabolites into and out of the mitochondria while excluding macromolecules. The IMM is a highly selective barrier between the solutes of the cytosol and those within the mitochondrial matrix. Cristae specialize in oxidative phosphorylation. The functions of these membranes are afforded by membrane proteins that are able to transport specific solutes. The appropriate localization, assembly into multi-subunit protein complexes, and wild-type function of these membrane proteins therefore is vital for mitochondria to maintain appropriate function and support cellular survival. This review will address the composition and functions of mitochondrial membrane localized multi-subunit protein complexes along with how these proteins undergo degradation to maintain homeostatic functions of mitochondria in the context of mitochondria specific transporters and ion channels. Due to the large number of known mitochondrial membrane transporters and ion channels this review will focus on the topics presented at the Mitochondrial Ion Channels and Transporters Symposium hosted by the New York University College of Dentistry in September 2015 in honor of Casey Kinnally.     

Stable ribosome binding to the endoplasmic reticulum enables compartment-specific regulation of mRNA translation

In eukaryotic cells, protein synthesis is compartmentalized; mRNAs encoding secretory/membrane proteins are translated on endoplasmic reticulum (ER)-bound ribosomes, whereas mRNAs encoding cytosolic proteins are translated on free ribosomes. mRNA partitioning between the two compartments occurs via positive selection: free ribosomes engaged in the translation of signal sequence-encoding mRNAs are trafficked from the cytosol to the ER. After translation termination, ER-bound ribosomes are thought to dissociate, thereby completing a cycle of mRNA partitioning. At present, the physiological basis for termination-coupled ribosome release is unknown. To gain insight into this process, we examined ribosome and mRNA partitioning during the unfolded protein response, key elements of which include suppression of the initiation stage of protein synthesis and polyribosome breakdown. We report that unfolded protein response (UPR)-elicited polyribosome breakdown resulted in the continued association, rather than release, of ER-bound ribosomes. Under these conditions, mRNA translation in the cytosol was suppressed, whereas mRNA translation on the ER was sustained. Furthermore, mRNAs encoding key soluble stress proteins (XBP-1 and ATF-4) were translated primarily on ER-bound ribosomes. These studies demonstrate that ribosome release from the ER is termination independent and identify new and unexpected roles for the ER compartment in the translational response to induction of the unfolded protein response.     

Microtubule-associated proteins bind to 30 kDa and 60 kDa proteins of rat brain mitochondria: visualization by ligand blotting

Axonal transport of mitochondria is a microtubule-associated movement. Microtubule-mitochondria interactions were studied in vitro using organelles isolated from rat brain. Thanks to the ligand blotting method we were able to show two mitochondrial membrane proteins with apparent molecular masses of 30 kDa and 60 kDa that bind microtubule-associated proteins. The binding of the 30 kDa protein has an apparent Kd of 8 x 10(-8) M. Digitonin fractionation of mitochondria reveals a bimodal localization of the 30 kDa and the 60 kDa proteins within the outer membrane. The data suggest that these polypeptides could participate to the interactions observed in situ between microtubules and mitochondria.     

Flock house virus RNA polymerase is a transmembrane protein with amino-terminal sequences sufficient for mitochondrial localization and membrane insertion

Localization of RNA replication to intracellular membranes is a universal feature of positive-strand RNA viruses. Replication complexes of flock house virus (FHV), the best-studied alphanodavirus, are located on outer mitochondrial membranes in infected Drosophila melanogaster cells and are associated with the formation of membrane-bound spherules, similar to structures found for many other positive-strand RNA viruses. To further study FHV replication complex formation, we investigated the subcellular localization, membrane association, and membrane topology of protein A, the FHV RNA-dependent RNA polymerase, in the yeast Saccharomyces cerevisiae, a host able to support full FHV RNA replication and virion formation. Confocal immunofluorescence revealed that protein A localized to mitochondria in yeast, as in Drosophila cells, and that this mitochondrial localization was independent of viral RNA synthesis. Nycodenz gradient flotation and dissociation assays showed that protein A behaved as an integral membrane protein, a finding consistent with a predicted N-proximal transmembrane domain. Protease digestion and selective permeabilization after differential epitope tagging demonstrated that protein A was inserted into the outer mitochondrial membrane with the N terminus in the inner membrane space or matrix and that the C terminus was exposed to the cytoplasm. Flotation and immunofluorescence studies with deletion mutants indicated that the N-proximal region of protein A was important for both membrane association and mitochondrial localization. Gain-of-function studies with green fluorescent protein fusions demonstrated that the N-terminal 46 amino acids of protein A were sufficient for mitochondrial localization and membrane insertion. We conclude that protein A targets and anchors FHV RNA replication complexes to outer mitochondrial membranes, in part through an N-proximal mitochondrial localization signal and transmembrane domain.     

Sequences required for delivery and localization of the ADP/ATP translocator to the mitochondrial inner membrane.

The ADP/ATP translocator, a transmembrane protein of the mitochondrial inner membrane, is coded in Saccharomyces cerevisiae by the nuclear gene PET9. DNA sequence analysis of the PET9 gene showed that it encoded a protein of 309 amino acids which exhibited a high degree of homology with mitochondrial translocator proteins from other sources. This mitochondrial precursor, in contrast to many others, does not contain a transient presequence which has been shown to direct the posttranslational localization of proteins in the organelle. Gene fusions between the PET9 gene and the gene encoding beta-galactosidase (lacZ) were constructed to define the location of sequences necessary for the mitochondrial delivery of the ADP/ATP translocator protein in vivo. These studies reveal that the information to target the hybrid molecule to the mitochondria is present within the first 115 residues of the protein. In addition, these studies suggest that the "import information" of the amino-terminal region of the ADP/ATP translocator precursor is twofold. In addition to providing targeting function of the precursor to the organelle, these amino-terminal sequences act to prevent membrane-anchoring sequences located between residues 78 and 98 from stopping import at the outer mitochondrial membrane. These results are discussed in light of the function of distinct protein elements at the amino terminus of mitochondrially destined precursors in both organelle delivery and correct membrane localization.     

Localization of mRNAs encoding human mitochondrial oxidative phosphorylation proteins

The mitochondrial oxidative phosphorylation (OXPHOS) proteins are encoded by both nuclear and mitochondrial DNA. The nuclear-encoded OXPHOS mRNAs have specific subcellular localizations, but little is known about which localize near mitochondria. Here, we compared mRNAs in mitochondria-bound polysome fractions with those in cytosolic, free polysome fractions. mRNAs encoding hydrophobic OXPHOS proteins, which insert into the inner membrane, were localized near mitochondria. Conversely, OXPHOS gene which mRNAs were predominantly localized in cytosol had less than one transmembrane domain. The RNA-binding protein Y-box binding protein-1 is localized at the mitochondrial outer membrane and bound to the OXPHOS mRNAs. Our findings offer new insight into mitochondrial co-translational import in human cells.     

The First Steps of Protein Import into Mitochondria

Protein import into mitochondria is initiated by the recognition and binding of precursor proteins by import components in the cytosol, on the mitochondrial surface, and in the mitochondrial outer membrane. Following their synthesis on cytoplasmic ribosomes, some precursor proteins interact with molecular chaperones in the cytosol which function in maintaining the precursor protein in an import-competent state and may also aid in the delivery of the precursor to the mitochondria. A multisubunit protein import receptor then recognises and binds precursor proteins before feeding them into the outer membrane import site. Some proteins are sorted from the import site into the outer membrane, but most precursor proteins travel through the outer membrane import site into the mitochondria, where the later steps of protein import take place.     

Protein import into mitochondria: ATP-dependent protein translocation activity in a submitochondrial fraction enriched in membrane contact sites and specific proteins

To identify the membrane regions through which yeast mitochondria import proteins from the cytoplasm, we have tagged these regions with two different partly translocated precursor proteins. One of these was bound to the mitochondrial surface of ATP-depleted mitochondria and could subsequently be chased into mitochondria upon addition of ATP. The other intermediate was irreversibly stuck across both mitochondrial membranes at protein import sites. Upon subfraction of the mitochondria, both intermediates cofractionated with membrane vesicles whose buoyant density was between that of inner and outer membranes. When these vesicles were prepared from mitochondria containing the chaseable intermediate, they internalized it upon addition of ATP. A non-hydrolyzable ATP analogue was inactive. This vesicle fraction contained closed, right-side-out inner membrane vesicles attached to leaky outer membrane vesicles. The vesicles contained the mitochondrial binding sites for cytoplasmic ribosomes and contained several mitochondrial proteins that were enriched relative to markers of inner or outer membranes. By immunoelectron microscopy, two of these proteins were concentrated at sites where mitochondrial inner and outer membranes are closely apposed. We conclude that these vesicles contain contact sites between the two mitochondrial membranes, that these sites are the entry point for proteins into mitochondria, and that the isolated vesicles are still translocation competent.     

Lateral release of proteins from the TOM complex into the outer membrane of mitochondria

The TOM complex of the outer membrane of mitochondria is the entry gate for the vast majority of precursor proteins that are imported into the mitochondria. It is made up by receptors and a protein conducting channel. Although precursor proteins of all subcompartments of mitochondria use the TOM complex, it is not known whether its channel can only mediate passage across the outer membrane or also lateral release into the outer membrane. To study this, we have generated fusion proteins of GFP and Tim23 which are inserted into the inner membrane and, at the same time, are spanning either the TOM complex or are integrated into the outer membrane. Our results demonstrate that the TOM complex, depending on sequence determinants in the precursors, can act both as a protein conducting pore and as an insertase mediating lateral release into the outer membrane.     

Coronavirus multiplication: locations of genes for virion proteins on the avian infectious bronchitis virus genome.

Six overlapping viral RNAs are synthesized in cells infected with the avian coronavirus infectious bronchitis virus (IBV). These RNAs contain a 3'-coterminal nested sequence set and were assumed to be viral mRNAs. The seven major IBV virion proteins are all produced by processing of three polypeptides of ca. 23, 51, and 115 kilodaltons. These are the core polypeptides of the small membrane proteins, the nucleocapsid protein, and the 155-kilodalton precursor to the large membrane proteins GP90 and GP84, respectively. To determine which mRNAs specify these polypeptides, we isolated RNA from infected cells and translated it in a messenger-dependent rabbit reticulocyte lysate. Proteins of 23, 51, and 110 kilodaltons were produced. Two-dimensional tryptic peptide mapping demonstrated that these proteins were closely related to the major virion proteins. Fractionation of the RNA before cell-free translation permitted the correlation of messenger activities for synthesis of the proteins with the presence of specific mRNAs. We found that the smallest RNA, RNA A, directs the synthesis of P51, the nucleocapsid protein. RNA C, which contains the sequences of RNA A, directs the synthesis of the small membrane protein P23. RNA E directs the synthesis of the large virion glycoproteins. These results supported a model in which only the unique 5'-terminal domain of each IBV mRNA is active in translation and enabled us to localize genes for virion proteins on the IBV genome.     

The C-terminal region of TIM17 links the outer and inner mitochondrial membranes in Arabidopsis and is essential for protein import

The translocase of the inner membrane 17 (AtTIM17-2) protein from Arabidopsis has been shown to link the outer and inner mitochondrial membranes. This was demonstrated by several approaches: (i) In vitro organelle import assays indicated the imported AtTIM17-2 protein remained protease accessible in the outer membrane when inserted into the inner membrane. (ii) N-terminal and C-terminal tagging indicated that it was the C-terminal region that was located in the outer membrane. (iii) Antibodies raised to the C-terminal 100 amino acids recognize a 31-kDa protein from purified mitochondria, but cross-reactivity was abolished when mitochondria were protease-treated to remove outer membrane-exposed proteins. Antibodies to AtTIM17-2 inhibited import of proteins via the general import pathway into outer membrane-ruptured mitochondria, but did not inhibit protein import via the carrier import pathway. Together these results indicate that the C-terminal region of AtTIM17-2 is exposed on the outer surface of the outer membrane, and the C-terminal region is essential for protein import into mitochondria.     

The relationship of protein and lipid synthesis during the biogenesis of mitochondrial membranes

Summary Rat liver mitochondria were fractionated into inner and outer membrane components at various times after the intravenous injection of¹⁴C-leucine or¹⁴C-glycerol. The time curves of protein and lecithin labeling were similar in the intact mitochondria, the outer membrane fraction, and the inner membrane fraction. In rat liver slices also, the kinetics of³H-phenylalanine incorporation into mitochondrial KCl-insoluble proteins was identical to that of¹⁴C-glycerol incorporation into mitochondrial lecithin. These results suggest a simultaneous assembly of protein and lecithin during membrane biogenesisThe proteins and lecithin of the outer membrane were maximally labeledin vivo within 5 min after injection of the radioactive precursors, whereas the insoluble proteins and lecithin of the inner membrane reached a maximum specific acitivity 10 min after injection.Phospholipid incorporation into mitochondria of rat liver slices was not affected when protein synthesis was blocked by cycloheximide, puromycin, or actinomycin D. The injection of cycloheximide 3 to 30 min prior to¹⁴C-choline did not affect thein vivo incorporation of lecithin into the mitochondrial inner or outer membranes; however treatment with the drug for 60 min prior to¹⁴C-choline resulted in a decrease in lecithin labeling. These results suggest that phospholipid incorporation into membranes may be regulated by the amount of newly synthesized protein available.When mitochondria and microsomes containing labeled phospholipids were incubated with the opposite unlabeled fractionin vitro, a rapid exchange of phospholipid between the microsomes and the outer membrane occurred. A slight exchange with the inner membrane was observed.     

Mitochondrial localization of AtOXA1, an arabidopsis homologue of yeast Oxa1p involved in the insertion and assembly of protein complexes in mitochondrial inner membrane

Components of some protein complexes present in the inner membrane of mitochondria are encoded in both nuclear and mitochondrial genomes, and correct sorting and assembly of these proteins is necessary for proper respiratory function. Recent studies in yeast suggest that Oxa1p, a protein conserved between prokaryotes and eukaryotes, is an essential factor for protein sorting and assembly into membranes. We previously identified AtOXA1, an Arabidopsis homologue of OXA1 by functional complementation of a yeast oxa1- mutant. In this study, we investigated the genomic organization of AtOXA1 and localization of the AtOXA1 protein. Characterization of the AtOXA1 genomic region indicated that the gene consists of 10 exons and is located on chromosome V. A database search also revealed another gene coding for a putative protein homologous to AtOXA1 on chromosome II. Transient expression of a green fluorescent protein (GFP) fusion in suspension-cultured tobacco cells showed that AtOXA1 is targeted into mitochondria by its N-terminal presequence. Antibodies raised against AtOXA1 recognized a 38-kDa intrinsic protein of the inner mitochondrial membrane. Thus, localization of AtOXA1 in the mitochondrial inner membrane, together with our previous complementation experiment in yeast, suggested that it is a functional homologue of Oxa1p.     

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

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

Calcium depletion and calmodulin inhibition affect the import of nuclear-encoded proteins into plant mitochondria

Many metabolic processes essential for plant viability take place in mitochondria. Therefore, mitochondrial function has to be carefully balanced in accordance with the developmental stage and metabolic requirements of the cell. One way to adapt organellar function is the alteration of protein composition. Since most mitochondrial proteins are nuclear encoded, fine-tuning of mitochondrial protein content could be achieved by the regulation of protein translocation. Here we present evidence that the import of nuclear-encoded mitochondrial proteins into plant mitochondria is influenced by calcium and calmodulin. In pea mitochondria, the calmodulin inhibitor ophiobolin A as well as the calcium ionophores A23187 and ionomycin inhibit translocation of nuclear-encoded proteins in a concentration-dependent manner, an effect that can be countered by the addition of external calmodulin or calcium, respectively. Inhibition was observed exclusively for proteins translocating into or across the inner membrane but not for proteins residing in the outer membrane or the intermembrane space. Ophiobolin A and the calcium ionophores further inhibit translocation into mitochondria with disrupted outer membranes, but their effect is not mediated via a change in the membrane potential across the inner mitochondrial membrane. Together, our results suggest that calcium/calmodulin influences the import of a subset of mitochondrial proteins at the inner membrane. Interestingly, we could not observe any influence of ophiobolin A or the calcium ionophores on protein translocation into mitochondria of yeast, indicating that the effect of calcium/calmodulin on mitochondrial protein import might be a plant-specific trait.