RESUMEN
The yeast cytosol contains multiple homologs of the DnaK and DnaJ chaperone family. Our current understanding of which homologs functionally interact is incomplete. Zuotin is a DnaJ homolog bound to the yeast ribosome. We have now identified the DnaK homolog Ssz1p/Pdr13p as zuotin's partner chaperone. Zuotin and Ssz1p form a ribosome-associated complex (RAC) that is bound to the ribosome via the zuotin subunit. RAC is unique among the eukaryotic DnaK-DnaJ systems, as the 1:1 complex is stable, even in the presence of ATP or ADP. In vitro, RAC stimulates the translocation of a ribosome-bound mitochondrial precursor protein into mitochondria, providing evidence for its chaperone-like effect on nascent chains. In agreement with the existence of a functional complex, deletion of each RAC subunit resulted in a similar phenotype in vivo. However, overexpression of zuotin partly rescued the growth defect of the Delta ssz1 strain, whereas overexpression of Ssz1p did not affect the Delta zuo1 strain, suggesting a pivotal function for the DnaJ homolog.
Asunto(s)
Proteínas de Unión al ADN/análisis , Proteínas de Unión al ADN/aislamiento & purificación , Proteínas de Escherichia coli , Proteínas Fúngicas/análisis , Proteínas Fúngicas/aislamiento & purificación , Chaperonas Moleculares/análisis , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/química , Citosol/fisiología , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/fisiología , Dimerización , Proteínas Fúngicas/química , Proteínas Fúngicas/fisiología , Proteínas del Choque Térmico HSP40 , Proteínas HSP70 de Choque Térmico/química , Proteínas de Choque Térmico/química , Mitocondrias , Chaperonas Moleculares/fisiología , Saccharomyces cerevisiae/fisiologíaRESUMEN
A class of integral membrane proteins, referred to as 'tail-anchored proteins', are inserted into phospholipid bilayers via a single segment of hydrophobic amino acids at the C-terminus, thereby displaying a large functional domain in the cytosol. This membrane attachment strategy allows eukaryotic cells to position a wide range of cytoplasmic activities close to the surface of an intracellular membrane. Tail-anchored proteins often, but not always, demonstrate a selective distribution to specific intracellular organelles. This membrane-specific distribution is required for the large number of targeting proteins that are tail-anchored, but may or may not be critical for the numerous tail-anchored pro-apoptotic and anti-apoptotic proteins of the Bcl-2 family. Recent work has begun to address the mechanism for targeting tail-anchored proteins to their resident membranes, but questions remain. What targeting signals determine each protein's intracellular location? Are there receptors for these signals and, if so, how do they function? What steps are required to integrate tail-anchored proteins into the phospholipid bilayers? In this Traffic interchange, we summarise what is known about tail-anchored proteins, and outline the areas that are currently under study.
Asunto(s)
Membranas Intracelulares/química , Proteínas de la Membrana/metabolismo , Estructura Terciaria de Proteína , Transporte de Proteínas/fisiología , Retículo Endoplásmico/metabolismo , Membranas Intracelulares/metabolismo , Cinética , Mitocondrias/metabolismo , Pliegue de ProteínaRESUMEN
In the evolution of mitochondria and plastids from endosymbiotic bacteria, most of the proteins that make up these organelles have become encoded by nuclear genes and must therefore be transported across the organellar membranes, following synthesis in the cytosol. The core component of the protein translocation machines in both the mitochondrial and plastid outer membranes appears to be a beta-barrel protein, perhaps a relic from their bacterial ancestry, distinguishing these translocases from the alpha-helical-based protein translocation pores found in all other eukaryotic membranes.
Asunto(s)
Proteínas de Escherichia coli , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Proteínas de Transporte de Membrana , Mitocondrias/metabolismo , Proteínas de Plantas , Plastidios/metabolismo , Proteínas de Saccharomyces cerevisiae , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Membranas Intracelulares/metabolismo , Proteínas de Transporte de Membrana Mitocondrial , Porinas/metabolismo , Conformación Proteica , Precursores de Proteínas/metabolismo , Transporte de Proteínas , Canales de Translocación SEC , Canales Aniónicos Dependientes del VoltajeRESUMEN
Transcription-induced recombination has been reported in all organisms from bacteria to mammals. We have shown previously that the yeast genes HPR1 and THO2 may be keys to the understanding of transcription-associated recombination, as they both affect transcription elongation and hyper-recombination in a concerted manner. Using a yeast strain that has the wild-type THO2 gene replaced by one encoding a His(6)-HA-tagged version, we have isolated an oligomeric complex containing four proteins: Tho2, Hpr1, Mft1 and a novel protein that we have named Thp2. We have reciprocally identified a complex containing Hpr1, Tho2 and Mft1 using anti-Mft1 antibodies in immunoprecipitation experiments. The protein complex is mainly nuclear; therefore, Tho2 and Hpr1 are physically associated. Like hpr1Delta and tho2Delta cells, mft1Delta and thp2Delta cells show mitotic hyper- recombination and impaired transcription elongation, in particular, through the bacterial lacZ sequence. Hyper-recombination conferred by mft1Delta and thp2Delta is only observed in DNA regions under transcription conditions. We propose that this protein complex acts as a functional unit connecting transcription elongation with the incidence of mitotic recombination.
Asunto(s)
Proteínas de Unión al ADN , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Animales , Secuencia de Bases , Cartilla de ADN/genética , Proteínas Fúngicas/genética , Expresión Génica , Genes Fúngicos , Sustancias Macromoleculares , Mitosis , Mutación , Proteínas Nucleares , Fenotipo , Recombinación Genética , Saccharomyces cerevisiae/citología , Fracciones Subcelulares/metabolismo , Temperatura , Factores de Transcripción/química , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transcripción GenéticaRESUMEN
A clear picture has emerged over the past years on how a 'classic' mitochondrial protein, like subunit IV of cytochrome c oxidase, might be targeted to mitochondria. The targeting and subsequent import process involves the commitment of the TOM (translocase in the outer mitochondrial membrane) receptor complex on the mitochondrial surface, a TIM (translocase in the inner mitochondrial membrane) translocation complex in the mitochondrial inner membrane, and assorted chaperones and processing enzymes within the organelle. Recent work suggests that while very many mitochondrial precursor proteins might follow this basic targeting pathway, a large number have further requirements if they are to be successfully imported. These include ribosome-associated factors and soluble factors in the cytosol, soluble factors in the mitochondrial intermembrane space, an additional TIM translocase in the inner membrane and a range of narrow specificity assembly factors in the inner membrane. This review is focused on the targeting of proteins up to the stage at which they enter the TOM complex in the outer membrane.
Asunto(s)
Proteínas Portadoras/metabolismo , Membranas Intracelulares/metabolismo , Proteínas de la Membrana/metabolismo , Mitocondrias/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas Portadoras/genética , Proteínas de la Membrana/genética , Proteínas/metabolismo , Ribosomas/metabolismo , Saccharomyces cerevisiae/genéticaAsunto(s)
Proteínas de Arabidopsis , Arabidopsis/genética , Cloroplastos/metabolismo , Proteínas Fúngicas/genética , Proteínas de la Membrana/genética , Proteínas de Transporte de Membrana , Mitocondrias/metabolismo , Proteínas de Plantas/genética , Receptores de Superficie Celular , Secuencia de Aminoácidos , Arabidopsis/metabolismo , Transporte Biológico , Compartimento Celular/fisiología , Proteínas Fúngicas/metabolismo , Proteínas de la Membrana/metabolismo , Datos de Secuencia Molecular , Proteínas de Plantas/metabolismoRESUMEN
In all eukaryotic organisms, messenger RNA (mRNA) is synthesized in the nucleus and then exported to the cytoplasm for translation. The export reaction requires the concerted action of a large number of protein components, including a set of shuttle proteins that can exit and re-enter the nucleus through the nuclear pore complex. Here, we show that, in Saccharomyces cerevisiae, the shuttle protein Npl3p leaves the nuclear pore complex entirely and continues to function in the cytoplasm. A mutation at position 219 in its RNA-binding domain leaves Npl3p lingering in the cytoplasm associated with polysomes. Yeast cells expressing the mutant Npl3(L-219S) protein show alterations in mRNA stability that can affect protein synthesis. As a result, defects in nascent polypeptide targeting to subcellular compartments such as the mitochondria are also suppressed.
Asunto(s)
Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Mitocondrias/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Biosíntesis de Proteínas , ARN Mensajero/metabolismo , Proteínas de Saccharomyces cerevisiae , Sitios de Unión , Transporte Biológico , Citoplasma/metabolismo , Mitocondrias/genética , Mutación , Estabilidad del ARN , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Ribosomas/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMEN
A homolog of the bacterial cell division gene ftsZ was isolated from the alga Mallomonas splendens. The nuclear-encoded protein (MsFtsZ-mt) was closely related to FtsZs of the alpha-proteobacteria, possessed a mitochondrial targeting signal, and localized in a pattern consistent with a role in mitochondrial division. Although FtsZs are known to act in the division of chloroplasts, MsFtsZ-mt appears to be a mitochondrial FtsZ and may represent a mitochondrial division protein.
Asunto(s)
Eucariontes/química , Proteínas de Unión al GTP/química , Proteínas de Unión al GTP/metabolismo , Mitocondrias/química , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Proteínas de Saccharomyces cerevisiae , Alphaproteobacteria/química , Proteínas de Arabidopsis , Evolución Biológica , Cloroplastos/química , Cloroplastos/fisiología , Eucariontes/genética , Eucariontes/fisiología , Eucariontes/ultraestructura , Proteínas Fúngicas/análisis , GTP Fosfohidrolasas/análisis , Proteínas de Unión al GTP/genética , Biblioteca de Genes , Microscopía Confocal , Microscopía Fluorescente , Mitocondrias/metabolismo , Mitocondrias/fisiología , Mitocondrias/ultraestructura , Proteínas Mitocondriales , Datos de Secuencia Molecular , Filogenia , Proteínas de Plantas/genética , Proteínas Recombinantes de Fusión/análisis , Saccharomyces cerevisiae/químicaAsunto(s)
Proteínas Fúngicas/química , Membranas Intracelulares/química , Proteínas de la Membrana/química , Translocasas Mitocondriales de ADP y ATP/química , Proteínas Bacterianas/química , Proteínas Fúngicas/metabolismo , Membranas Intracelulares/enzimología , Proteínas de la Membrana/metabolismoRESUMEN
Inhibitors of apoptosis (IAPs) are a family of proteins that bear baculoviral IAP repeats (BIRs) and regulate apoptosis in vertebrates and Drosophila melanogaster. The yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe both encode a single IAP, designated BIR1 and bir1, respectively, each of which bears two BIRs. In rich medium, BIR1 mutant S. cerevisiae underwent normal vegetative growth and mitosis. Under starvation conditions, however, BIR1 mutant diploids formed spores inefficiently, instead undergoing pseudohyphal differentiation. Most spores that did form failed to survive beyond two divisions after germination. bir1 mutant S. pombe spores also died in the early divisions after spore germination and became blocked at the metaphase/anaphase transition because of an inability to elongate their mitotic spindle. Rather than inhibiting caspase-mediated cell death, yeast IAP proteins have roles in cell division and appear to act in a similar way to the IAPs from Caenorhabditis elegans and the mammalian IAP Survivin.
Asunto(s)
Proteínas Fúngicas/metabolismo , Saccharomyces cerevisiae/fisiología , Schizosaccharomyces/fisiología , Secuencia de Aminoácidos , Animales , División Celular/fisiología , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Humanos , Meiosis , Microscopía Electrónica , Datos de Secuencia Molecular , Secuencias Repetitivas de Aminoácido , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/genética , Schizosaccharomyces/citología , Schizosaccharomyces/genética , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Esporas Fúngicas , Vacuolas/fisiología , Vacuolas/ultraestructuraRESUMEN
Tail-anchored proteins are inserted into intracellular membranes via a C-terminal transmembrane domain. The topology of the protein is such that insertion must occur post-translationally, since the insertion sequence is not available for membrane insertion until after translation of the tail-anchored polypeptide is completed. Here, we show that the targeting information in one such tail-anchored protein, translocase in the outer mitochondrial membrane 22, is contained in a short region flanking the transmembrane domain. An equivalent region is sufficient to specify the localisation of Bcl2 and SNARE proteins to the secretory membranes. We discuss the targeting process for directing members of this protein family to the secretory and mitochondrial membranes in vivo.
Asunto(s)
Proteínas Fúngicas/metabolismo , Proteínas de la Membrana/metabolismo , Mitocondrias/metabolismo , Saccharomyces cerevisiae/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Transporte Biológico , Proteínas Portadoras/metabolismo , Datos de Secuencia Molecular , Saccharomyces cerevisiae/ultraestructuraRESUMEN
The targeting of newly-made polypeptides to specific membranes, and the subsequent ability of a membrane to allow only certain polypeptides into its compartment, are essential to maintain the ultrastructure of Eukaryotic cells. Distinct oligomeric protein complexes in each cellular membrane catalyse these translocation processes. A recent report [Hill K et al. Nature 1998;395:516-521 (Ref. 1)] of the reconstitution of the translocation channel from the mitochondrial outer membrane, after producing the major structural component of the channel by recombinant means, promises a system to dissect in molecular detail the exact working of one of these protein translocation machines.
Asunto(s)
Proteínas Portadoras/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Mitocondrias/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Bacterias/metabolismo , Membranas Intracelulares/metabolismo , Proteínas de Transporte de Membrana Mitocondrial , Proteínas del Complejo de Importación de Proteínas Precursoras Mitocondriales , Proteínas Mitocondriales/biosíntesis , Neurospora crassa/metabolismo , Neurospora crassa/ultraestructura , Porinas/metabolismoRESUMEN
When overexpressed in Saccharomyces cerevisiae, beta-galactosidase fusion proteins directed to the mitochondria are toxic, preventing growth of yeast cells on non-fermentable carbon sources (Emr, S. D., Vassarotti, A., Garrett, J., Geller, B. L., Takeda, M., and Douglas, M. G. (1986) J. Cell Biol. 102, 523-533). We show that such fusion proteins interfere with the assembly of respiratory complexes in the mitochondrial inner membrane, without blocking protein translocation. The gene YME1, encoding an ATP-dependent metalloprotease of the mitochondrial inner membrane, acts as a suppressor of this defect; a 3-fold overexpression of Yme1p is sufficient to restore respiratory complex assembly and mitochondrial function. Detailed knowledge of the topology and effect of the toxic beta-galactosidase fusion proteins will permit the identification and characterization of components that control protein sorting and protein assembly within the mitochondrial inner membrane.
Asunto(s)
Membranas Intracelulares/efectos de los fármacos , Mitocondrias/efectos de los fármacos , ATPasas de Translocación de Protón/toxicidad , Proteínas Recombinantes de Fusión/toxicidad , Proteínas de Saccharomyces cerevisiae , beta-Galactosidasa/toxicidad , Proteasas ATP-Dependientes , Adenosina Trifosfatasas , Transporte Biológico , Proteínas Fúngicas/biosíntesis , Sustancias Macromoleculares , Consumo de Oxígeno/efectos de los fármacos , ATPasas de Translocación de Protón/genética , Saccharomyces cerevisiae , Supresión Genética , beta-Galactosidasa/genéticaRESUMEN
The yeast nascent polypeptide-associated complex (NAC) is encoded by two genes, EGD1 and EGD2, and is associated with cytoplasmic ribosomes. Yeast mutants lacking NAC (Deltaegd2) are viable but suffer slight defects in the targeting of nascent polypeptides to several locations including the endoplasmic reticulum and mitochondria. If both NAC and Mft52p are missing from yeast cells, inefficient targeting of mitochondrial precursor proteins leads to defects in both mitochondrial function and morphology. We suggest that NAC provides a ribosomal environment for nascent mitochondrial targeting sequences to achieve secondary structure, thereby enhancing the efficiency of protein targeting.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Proteínas Fúngicas/metabolismo , Mitocondrias/metabolismo , Procesamiento Proteico-Postraduccional , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Citosol/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Eliminación de Gen , Modelos Biológicos , Datos de Secuencia Molecular , Proteínas Recombinantes de Fusión/biosíntesis , Ribosomas/metabolismo , Alineación de Secuencia , Factores de Transcripción/química , Factores de Transcripción/genética , beta-Galactosidasa/biosíntesisRESUMEN
Yeast cells harboring mft1 mutations are compromised in mitochondrial protein targeting, and Mft1p has previously been identified as a ribosomal protein. However, two genes, PLC2 and YML062C, are present in the MFT1 locus, and we show that mft1 mutant cells are compromised in the function of the cytosolic protein encoded by YML062C. The ribosomal protein (YS3a) is actually encoded by the tightly linked PLC2 gene, and does not play a role in targeting proteins to the mitochondria.
Asunto(s)
Compartimento Celular , Proteínas de Unión al ADN , Proteínas Fúngicas/genética , Genes Fúngicos , Mitocondrias/metabolismo , Precursores de Proteínas/metabolismo , Proteínas de Saccharomyces cerevisiae , Levaduras/genética , Secuencia de Aminoácidos , Ligamiento Genético , Datos de Secuencia Molecular , Mutagénesis Insercional , Unión Proteica , Biosíntesis de Proteínas , Proteínas Ribosómicas/genética , Homología de Secuencia de AminoácidoRESUMEN
Messenger RNA (mRNA) localisation is one of the prime mechanisms to ensure protein localisation in the cytoplasm of polarised embryonic cells, and has been well-studied in the development of Xenopus and Drosophila embryos. But what of other cells? Here, we discuss whether the directed transport of mRNA out of the nucleus, following cytoplasmic highways to a specified organelle in the cytoplasm, might also contribute to the exquisite fidelity of protein targeting observed in all eukaryotic cells.
Asunto(s)
Mitocondrias/metabolismo , Proteínas/metabolismo , ARN Mensajero/metabolismo , Proteínas de Saccharomyces cerevisiae , Animales , Transporte Biológico , Núcleo Celular/genética , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Drosophila/genética , Drosophila/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Mamíferos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Biosíntesis de Proteínas , Procesamiento Proteico-Postraduccional , Proteínas/genética , ARN Mensajero/genética , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismoRESUMEN
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.
Asunto(s)
Mitocondrias/metabolismo , Proteínas/metabolismo , Sitios de Unión , Transporte Biológico , Chaperonas Moleculares , Receptores de Superficie Celular , RibosomasRESUMEN
We have identified a novel protein, Mft52, in the cytosol of yeast cells. Mft52 has a two-domain structure that includes a receptor-like carboxyl-terminal "acid-bristle" domain, which binds basic, amphipathic mitochondrial targeting sequences. Native Mft52, purified from the cytosol of yeast cells, is found as a large particle eluting in the void volume of a Superose 6 gel filtration column. Fusion proteins, consisting of mitochondrial targeting sequences fused to nonmitochondrial passenger proteins, are targeted to mitochondria in wild-type yeast cells, but defects in the gene encoding Mft52 drastically reduce the delivery of these proteins to the mitochondria. We propose that Mft52 is a subunit of a particle that is part of a system of targeting factors and molecular chaperones mediating the earliest stages of protein targeting to the mitochondria.
Asunto(s)
Proteínas Portadoras/aislamiento & purificación , Proteínas Fúngicas/aislamiento & purificación , Mitocondrias/metabolismo , Precursores de Proteínas/aislamiento & purificación , Saccharomyces cerevisiae/metabolismo , Secuencia de Aminoácidos , Transporte Biológico , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Citosol/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Datos de Secuencia Molecular , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismoRESUMEN
Protein import into yeast mitochondria is mediated by four integral outer membrane proteins which function as import receptors. These proteins (termed Mas20p, Mas22p, Mas37p and Mas70p) appear to exist as two subcomplexes: a Mas37p-Mas70p heterodimer and a less well characterized Mas20p-Mas22p complex. The subcomplexes interact functionally during protein import, but it has remained uncertain whether they are in direct contact with each other in vivo. Here we show that Mas20p and Mas70p can be cross-linked in intact mitochondria, or co-immunoprecipitated from digitonin-solubilized mitochondria. Furthermore, the cytosolic domains of these two proteins interact in the 'two-hybrid' system. Association of Mas20p and Mas70p is virtually abolished by a mutation in the single tetratricopeptide motif in Mas20p. This mutation specifically inhibits import of precursors that are first recognized by Mas37p-Mas70p and only then transferred to Mas20p-Mas22p. We conclude that the two receptor subcomplexes of the mitochondrial protein import receptor interact in vivo via their Mas20p and Mas70p subunits and that this interaction is functionally important.
Asunto(s)
Proteínas Fúngicas/metabolismo , Proteínas de la Membrana/metabolismo , Mitocondrias/metabolismo , Receptores Citoplasmáticos y Nucleares , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Secuencia de Bases , Transporte Biológico , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Mitocondrias/química , Mitocondrias/genética , Proteínas de Transporte de Membrana Mitocondrial , Proteínas del Complejo de Importación de Proteínas Precursoras Mitocondriales , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Unión Proteica , Conformación Proteica , Secuencias Repetitivas de Ácidos Nucleicos , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genéticaRESUMEN
Mitochondrial precursor proteins made in the cytosol bind to a hetero-oligomeric protein import receptor on the mitochondrial surface and then pass through the translocation channel across the outer membrane. This translocation step is accelerated by an acidic domain of the receptor subunit Mas22p, which protrudes into the intermembrane space. This 'trans' domain of Mas22p specifically binds functional mitochondrial targeting peptides with a Kd of < 1 microM and is required to anchor the N-terminal targeting sequence of a translocation-arrested precursor in the intermembrane space. If this Mas22p domain is deleted, respiration-driven growth of the cells is compromised and import of different precursors into isolated mitochondria is inhibited 3- to 8-fold. Binding of precursors to the mitochondrial surface appears to be mediated by cytosolically exposed acidic domains of the receptor subunits Mas20p and Mas22p. Translocation of a precursor across the outer membrane thus appears to involve sequential binding of the precursor's basic and amphiphilic targeting signal to acidic receptor domains on both sides of the membrane.