Import of proteins into mitochondria: nucleotide sequence of the gene for a 70-kd protein of the yeast mitochondrial outer membrane.

The nucleotide sequence of the yeast chromosomal gene coding for the 70-kd protein of the mitochondrial outer membrane was determined. The deduced amino acid sequence of the protein agrees with the experimentally determined size and amino acid composition of the purified protein and correctly predicts the fragments obtained by cleaving the protein at its single tryptophan residue. The deduced NH2-terminal sequence features an uninterrupted stretch of 28 uncharged amino acids flanked on both sides by basic amino acids. By sequencing a truncated version of the gene it was found that the corresponding polypeptide product lacks the 203 carboxy-terminal amino acids of the authentic 70-kd protein. As shown in the accompanying paper, this protein fragment still becomes attached to the mitochondrial outer membrane in vivo.     

Import of proteins into mitochondria: a 70 kilodalton outer membrane protein with a large carboxy-terminal deletion is still transported to the outer membrane.

The yeast mitochondrial outer membrane contains a major 70-kd protein which is coded by a nuclear gene. Two forms of this gene were isolated from a yeast genomic clone bank: the intact gene, and a truncated gene which had lost a large part of its 3' end during the cloning procedure. Upon transformation into yeast, both the intact and the truncated gene are expressed; the truncated gene generates a shortened protein missing 203 amino acids from the carboxy-terminus. This truncated polypeptide reacts with a monoclonal antibody against the authentic 70-kd protein and is transported to the mitochondrial outer membrane. By integrative transformation, we have constructed a yeast mutant which lacks the 70-kd protein and is unable to adapt to growth on a nonfermentable carbon source at 37 degrees C. This phenotypic lesion can be corrected by transforming the mutant with the intact, but not the truncated gene. The carboxy-terminal sequence of 203 amino acids is thus necessary for the function of the protein, but not for its targeting to the mitochondrion.     

The yeast ATP synthase subunit 4: structure and function

The structure of ATP synthase subunit 4 was determined by using the oligonucleotide probe procedure. This subunit is the fourth polypeptide of the complex when classifying subunits in order of decreasing molecular mass. Its relative molecular mass is 25 kDa. The ATP4 gene was isolated and sequenced. The nucleotide sequence predicts that subunit 4 is probably derived from a precursor protein 244 amino acids long. Mature subunit 4 contains 209 amino acid residues and the predicted molecular mass is 23250 kDa. Subunit 4 shows homology with the b-subunit of Escherichia coli ATP synthase and the b-subunit of beef heart mitochondrial ATP synthase. By using homologous transformation, a mutant lacking wild subunit 4 was constructed. This mutant is devoid of oxidative phosphorylation and F1 is loosely bound to the membrane. Our data are in favor of a structural relationship between subunit 4 and the mitochondrially-translated subunit 6 during biogenesis of F0.     

The first twelve amino acids of a yeast mitochondrial outer membrane protein can direct a nuclear-coded cytochrome oxidase subunit to the mitochondrial inner membrane.

We have used an in vivo complementation assay to test whether a given polypeptide sequence can direct an attached protein to the mitochondrial inner membrane. The host is a previously described yeast deletion mutant that lacks cytochrome oxidase subunit IV (an imported protein) and, thus neither assembles cytochrome oxidase in its mitochondrial inner membrane nor grows on the non-fermentable carbon source, glycerol. Growth on glycerol and cytochrome oxidase assembly are restored to the mutant if it is transformed with the gene encoding authentic subunit IV precursor, a protein carrying a 25-residue transient pre-sequence. No restoration is seen with a plasmid encoding a subunit IV precursor whose pre-sequence has been shortened to seven residues. Partial, but significant restoration is achieved by an artificial subunit IV precursor in which the authentic pre-sequence has been replaced by the first 12 amino acids of a 70-kd protein of the mitochondrial outer membrane. If this dodecapeptide is fused to the amino terminus of mouse dihydrofolate reductase (a cytosolic protein), the resulting fusion protein is imported into the matrix of yeast mitochondria in vitro and in vivo. Import in vitro requires an energized inner membrane. We conclude that the extreme amino terminus of the 70-kd outer membrane protein can direct an attached protein across the mitochondrial inner membrane.     

A dual role for mitochondrial heat shock protein 70 in membrane translocation of preproteins

The role of mitochondrial 70-kD heat shock protein (mt-hsp70) in protein translocation across both the outer and inner mitochondrial membranes was studied using two temperature-sensitive yeast mutants. The degree of polypeptide translocation into the matrix of mutant mitochondria was analyzed using a matrix-targeted preprotein that was cleaved twice by the processing peptidase. A short amino-terminal segment of the preprotein (40-60 amino acids) was driven into the matrix by the membrane potential, independent of hsp70 function, allowing a single cleavage of the presequence. Artificial unfolding of the preprotein allowed complete translocation into the matrix in the case where mutant mt-hsp70 had detectable binding activity. However, in the mutant mitochondria in which binding to mt-hsp70 could not be detected the mature part of the preprotein was only translocated to the intermembrane space. We propose that mt-hsp70 fulfills a dual role in membrane translocation of preproteins. (a) Mt-hsp70 facilitates unfolding of the polypeptide chain for translocation across the mitochondrial membranes. (b) Binding of mt-hsp70 to the polypeptide chain is essential for driving the completion of transport of a matrix- targeted preprotein across the inner membrane. This second role is independent of the folding state of the preprotein, thus identifying mt- hsp70 as a genuine component of the inner membrane translocation machinery. Furthermore we determined the sites of the mutations and show that both a functional ATPase domain and ATP are needed for mt- hsp70 to bind to the polypeptide chain and drive its translocation into the matrix.     

Biogenesis of mitochondria: DNA sequence analysis of mit- mutations in the mitochondrial oli1 gene coding for mitochondrial ATPase subunit 9 in Saccharomyces cerevisiae.

The nucleotide sequence of the yeast mitochondrial olil gene has been obtained in a series of mit- mutants with mutations in this gene, which codes for subunit 9 of of the mitochondrial ATPase complex. Subunit 9 is the proteolipid, 76 amino acids in length, necessary for the proton translocation function of the membrane Fo-sector. These mutants were classified on the basis of their rescue by a petite strain shown here to retain the entire wild-type olil gene. The mutation in one mit- strain removes a positively charged residue (Arg39----Met) which is likely to be located in a segment of subunit 9 that protrudes from the inner mitochondrial membrane. In a second mit- mutant, a negatively charged residue replaces a conserved glycine residue (Gly18----Asp) in a glycine-rich segment of the protein that is most likely embedded within the membrane. Other mit- mutations result in frameshifts with predicted products 7, 65 and 68 amino acid residues long. In each mit- mutant, there is the loss of one or more of the amino acid residues that are highly conserved among diverse species. The location and nature of specific changes pinpoint amino acid residues in subunit 9 essential to the activity of the mitochondrial ATPase complex.     

Transport of the yeast ATP synthase beta-subunit into mitochondria. Effects of amino acid substitutions on targeting.

We have isolated the yeast ATP2 gene encoding the beta-subunit of mitochondrial ATP synthase and determined its nucleotide sequence. A fusion between the N-terminal 15 amino acid residues of beta-subunit and the mouse cytosolic protein dihydrofolate reductase (DHFR) was transcribed and translated in vitro and found to be transported into isolated yeast mitochondria. A fusion with the first 35 amino acid residues of beta-subunit attached to DHFR was not only transported but also proteolytically processed by a mitochondrial protease. Amino acid substitutions were introduced into the N-terminal presequence of the beta-subunit by bisulphite mutagenesis of the corresponding DNA. The effects of these mutations on mitochondrial targeting were assessed by transport experiments in vitro using DHFR fusion proteins. All of the mutants, harbourin from one to six amino acid substitutions in the first 14 residues of the presequence, were transported into mitochondria, though at least one of them (I8) was transported and proteolytically processed at a much reduced rate. The I8 mutant beta-subunit also exhibited poor transport and processing in vivo, and expression of this mutant polypeptide failed to complement the glycerol- phenotype of a yeast ATP2 mutant. More remarkably, the expression of I8 beta-subunit induced a more general growth defect in yeast, possibly due to interference with the transport of other, essential, mitochondrial proteins.     

Mammalian multidrug resistance gene: complete cDNA sequence indicates strong homology to bacterial transport proteins

The complete nucleotide and primary structure (1276 amino acids) of a full length mdr cDNA capable of conferring a complete multidrug-resistant phenotype is presented. The deduced amino acid sequence suggests that mdr is a membrane glycoprotein which includes six pairs of transmembrane domains and a cluster of potentially N-linked glycosylation sites near the amino terminus. A striking feature of the protein is an internal duplication that includes approximately 500 amino acids. Each duplicated segment includes a consensus ATP-binding site. Amino acid homology is observed between the mdr gene and a series of bacterial transport genes. This strong homology suggests that a highly conserved functional unit involved in membrane transport is present in the mdr polypeptide. We propose that an energy-dependent transport mechanism is responsible for the multidrug-resistant phenotype.     

Neurospora endo-exonuclease is immunochemically related to the recC gene product of Escherichia coli.

Immunochemical cross-reaction between the endo-exonuclease of Neurospora crassa, an enzyme previously implicated in recombination and recombinational DNA repair, and the recC-encoded polypeptide of Escherichia coli has been detected by immunoblotting extracts of strains of E. coli having a deletion that includes the recBCD genes but carrying multicopy plasmids bearing all three of the recBCD genes or only one or two of these genes. It was predicted that homology would also be found at the amino acid sequence level between the recC polypeptide and both nuclear and mitochondrial endo-exonucleases of Saccharomyces cerevisiae, which cross-react with antibodies raised to the N. crassa endo-exonuclease. Since the gene for the S. cerevisiae mitochondrial enzyme, NUC1, has been cloned and sequenced and the predicted amino acid sequence is known, this sequence was aligned with the predicted amino acid sequence of the recC polypeptide. Extensive homology was found by aligning 306 of the 329 amino acids of the yeast mitochondrial nuclease sequence with the carboxy-terminal one-quarter of the amino acid sequence of the recC polypeptide.     

On the identification of the uncoupler binding protein of bovine mitochondrial oligomycin sensitive ATPase

The 55,000 dalton polypeptide component of the membrane sector of the mitochondrial oligomycin sensitive ATPase has been purified by recycling chromatography on BioGel P-100. The amino acid composition of the purified polypeptide differs significantly from that of the α-subunit of F₁ with which it shares a similar apparent molecular weight. However, the amino acid composition of the former is identical to that of the Factor B polypeptide, which is known to occur in oligomeric forms. Evidence is presented which suggests that the mitochondrial uncoupler binding proteins and the various oligomeric forms of the Factor B polypeptide, including the 55,000 dalton species described in the present report, are identical.     

The amino acid sequences of two 13 kDa polypeptides and partial amino acid sequence of 30 kDa polypeptide of complex I from bovine heart mitochondria: possible location of iron-sulfur clusters

Mitochondrial NADH:ubiquinone oxidoreductase (complex I) is the most complicated system in the respiratory chain. It consists of many subunits, some of which hold iron-sulfur clusters, but structural information is still limited. The amino acid sequences of two 13 kDa polypeptides, 13 kDa-A and 13 kDa-B polypeptides, of iron-sulfur protein fraction (IP) of bovine heart mitochondrial complex I were determined by a combination of protease digestion, Edman degradation, and carboxypeptidase digestion. The 13 kDa-A polypeptide was composed of 96 amino acids with a molecular weight of 10,536. The 13 kDa-B polypeptide consisted of 114 amino acids and had an acetylated amino terminus. The molecular weight of this protein was calculated to be 13,130 including the acetyl group. These proteins had no obvious sequence similarity to other known proteins. The partial amino acid sequence of 30 kDa-B polypeptide of IP was also determined to reveal a characteristic arrangement of cysteine residues that could be involved in iron-sulfur cluster formation.     

Identification of hydrophobic residues in the signal sequence of mitochondrial preornithine carbamyltransferase that enhance the rate of precursor import

Previous studies employing circular dichroism and resonance energy transfer techniques have demonstrated that the signal peptide of mitochondrial preornithine carbamyltransferase (pOCT) has the potential to interact with the surface of an anionic phospholipid membrane via a short amphiphilic helical domain. Here we have used predictive secondary structure computations as a guide to localize the putative membrane binding region in the pOCT signal sequence and demonstrate that replacement of leucine residues at positions 5, 8, and 9 with the less hydrophobic residue, alanine, significantly reduces the rate of precursor import (4-5-fold compared to wild type); the amino acid substitutions had little effect, however, on the ability of a mitochondrial matrix extract to process the mutant precursor polypeptide. The mutant precursor bound to anionic liposomes with a lower affinity compared to wild-type pOCT and was inhibited to a lesser extent than pOCT during import into mitochondria in the presence of varying concentrations of liposomes. Taken together, the results suggest that this small region of the pOCT signal sequence, containing a limited number of critical hydrophobic residues, contributes to the optimal rate of precursor import, perhaps by functioning as a membrane surface-seeking entity.     

Studies on the biosynthesis of protein by lactating guinea-pig mammary gland: Characteristics of the synthesis of α-lactalbumin and total protein by slices and cell-free systems

1. Slices of lactating guinea-pig mammary gland were incubated with radioactive amino acids and the various subcellular fractions separated by centrifugation after disruption of the cells by mincing and homogenization. The most active fraction for protein synthesis appeared to be the `mitochondrial'. 2. When the subcellular fractions were prepared without previous incubation of the cells and were then incubated with radioactive amino acid and an energy-generating system, the `mitochondrial fraction' was at least as active for protein synthesis as the `microsomal fraction'. 3. The ribosomes in the microsomal fraction are mainly unattached to membrane whereas those in the mitochondrial fraction are probably attached to fragments of the rough-surfaced endoplasmic reticulum. This latter fraction contains few mitochondria. 4. The combined mitochondrial and microsomal fractions incorporated radioactive amino acids into α-lactalbumin. 5. The radioactive leucine isolated from tryptic and chymotryptic peptides of α-lactalbumin synthesized in the cell-free system was not of uniform specific radioactivity. This was consistent with the polypeptide being assembled by the sequential addition of amino acids. 6. Evidence is presented for the polypeptide chain of α-lactalbumin being assembled from the N-terminus and for chain initiation in the cell-free system. 7. It is concluded that cell-free extracts of lactating mammary gland synthesize α-lactalbumin.     

Structural and functional studies of the amino terminus of yeast metallothionein

Purified yeast copper-metallothionein lacks 8 amino-terminal residues that are predicted from the DNA sequence of its gene. The removed sequence is unusual for metallothionein in its high content of hydrophobic and aromatic residues and its similarity to mitochondrial leader sequences. To study the significance of this amino-terminal cleavage, several mutations were introduced into the metallothionein coding gene, CUP1. One mutant, which deletes amino acid residues 2-8, had a minor effect on the ability of the molecule to confer copper resistance to yeast but did not affect CUP1 gene regulation. A second mutation, which changes two amino acids adjacent to the cleavage site, blocked removal of the extension peptide but had no effect on copper detoxification or gene regulation. Immunofluorescence studies showed that both the wild-type and these two mutant proteins are predominantly cytoplasmic with no evidence for mitochondrial localization. The cleavage site mutation allowed isolation and structural characterization of a full length metallothionein polypeptide. The copper content and luminescent properties of this molecule were identical to those of the truncated wild-type protein indicating a homologous cluster structure. Moreover, the amino-terminal peptide was selectively removed by various endopeptidases and an exopeptidase suggesting that it does not participate in the tertiary fold. These results argue that the amino-terminal peptide is not required for either the structural integrity or biological function of yeast metallothionein.     

Nucleotide sequence of ATPase subunit 6 gene of maize mitochondria

The ATPase subunit 6, located in the inner mitochondrial membrane, is encoded by mitochondrial genomes in animals and fungi. We have isolated and characterized a mitochondrial gene, designated atp 6, that encodes the subunit 6 polypeptide of Zea mays. Nucleotide and predicted amino acid sequence comparisons have revealed a homology of 44.6 and 33.2% with the yeast ATPase subunit 6 gene and polypeptide, respectively. The predicted protein in maize contains 291 amino acids with a molecular weight of 31,721. Hydropathy profiles generated for the maize and yeast polypeptides are very similar and contain large hydrophobic domains, characteristic of membrane bound proteins. RNA transfer blot analysis indicates that atp 6 is actively transcribed. Interestingly, 122 base pairs of nucleotide sequence interior to atp 6 have extensive homology with the 5′ end of the cytochrome oxidase subunit II gene of maize mitochondria, suggesting recombination between the two genes.     

A 70-kd protein of the yeast mitochondrial outer membrane is targeted and anchored via its extreme amino terminus

The major 70-kd protein of the yeast mitochondrial outer membrane is made on cytosolic ribosomes and imported into the outer membrane without proteolytic cleavage. We have attempted to identify the sequences which target the protein to the mitochondria and which permanently anchor it to the lipid bilayer of the outer membrane. By manipulating the cloned gene we have deleted 13 different regions throughout the polypeptide; in addition, we have fused amino-terminal regions of different length to beta-galactosidase. Each altered gene was introduced into yeast and the intracellular fate of the corresponding polypeptide product was determined by subcellular fractionation. All the information for targeting and anchoring the 70-kd protein (617 amino acids) was contained within the amino-terminal 41 amino acids. When this entire region was deleted, the protein was recovered with the cytosol fraction. However, several restricted deletions within this amino-terminal region appeared to affect targeting and anchoring differentially: most of the altered protein remained in the cytosol but a small fraction was misrouted into the mitochondrial matrix space. We suggest that targeting is mediated by a region which includes the 11 amino-terminal amino acids whereas the permanent membrane anchor is provided by a typical transmembrane sequence between residues 9 and 38.     

Study of the molecular organization of visual rhodopsin in photoreceptor membranes by limited proteolysis

Proteolysis of rhodopsin in disc membranes of right-side out orientation by thermolysin, papain and St. aureus V8 protease allowed to identify two highly exposed regions of polypeptide chain located on the cytoplasmic membrane surface: carboxyl terminal sequence 321-348 and the fragment 236-241. Incubation with chymotrypsin reveals the third site on the cytoplasmic surface, 146-147, accessible to proteolytic enzymes. Frozen-thawed membranes comprise a mixture of vesicles with normal and inverted orientation. Both thermolytic and chymotryptic digests of rhodopsin in these membranes contain the polypeptide which represents the amino terminal sequence lacking the first 30 amino acid residues. Thus at least 30 amino acids from the N-terminus must protrude into the intradiscal space. One additional site was located on the intradiscal surface: papain digests rhodopsin in the inverted membranes at the position 186-187. Localization of the proteolytic cleavage sites allowed to propose a model for rhodopsin topography in disc membrane: the polypeptide chain traverses the bilayer thickness seven times; each of seven transmembrane segments containing approximately 40 amino acid residues includes a sequence of approximately 30 hydrophobic amino acids; which are probably in close contact with hydrocarbon matrix of the membrane. Hydrophobic sequences are terminated with fragments containing clusters of hydrophilic amino acids, possibly interacting with lipid polar head groups and orienting each segment in the bilayer.     

Functional analysis of the Agrobacterium tumefaciens T-DNA transport pore protein VirB8

The VirB8 protein of Agrobacterium tumefaciens is essential for DNA transfer to plants. VirB8, a 237-residue polypeptide, is an integral membrane protein with a short N-terminal cytoplasmic domain. It interacts with two transport pore proteins, VirB9 and VirB10, in addition to itself. To study the role of these interactions in DNA transfer and to identify essential amino acids of VirB8, we introduced random mutations in virB8 by the mutagenic PCR method. The putative mutants were tested for VirB8 function by the ability to complement a virB8 deletion mutant in tumor formation assays. After multiple rounds of screening 13 mutants that failed to complement the virB8 deletion mutation were identified. Analysis of the mutant strains by DNA sequence analysis, Western blot assays, and reconstruction of new point mutations led to the identification of five amino acid residues that are essential for VirB8 function. The substitution of glycine-78 to serine, serine-87 to leucine, alanine-100 to valine, arginine-107 to proline or alanine, and threonine-192 to methionine led to the loss of VirB8 activity. When introduced into the wild-type strain, virB8(S87L) partially suppressed the tumor forming ability of the wild-type protein. Analysis of protein-protein interaction by the yeast two-hybrid assay indicated that VirB8(R107P) is defective in interactions with both VirB9 and VirB10. A second mutant VirB8(S87L) is defective in interaction with VirB9.     

Role of the carboxy terminus of polypeptide D1 in the assembly of a functional water-oxidizing manganese cluster in photosystem II of the cyanobacterium Synechocystis sp. PCC 6803: assembly requires a free carboxyl group at C-terminal position 344.

The D1 polypeptide of the photosystem II (PSII) reaction center is synthesized as a precursor polypeptide which is posttranslationally processed at the carboxy terminus. It has been shown in spinach that such processing removes nine amino acids, leaving Ala344 as the C-terminal residue [Takahashi, M., Shiraishi, T., & Asada, K. (1988) FEBS Lett. 240, 6-8; Takahashi, Y., Nakane, H., Kojima, H., & Satoh, K. (1990) Plant Cell Physiol. 31, 273-280]. We show here that processing on the carboxy side of Ala344 also occurs in the cyanobacterium Synechocystis 6803, resulting in the removal of 16 amino acids. By constructing a deletion strain of Synechocystis 6803 that lacks the three copies of the psbA gene encoding D1, we have developed a system for generating psbA mutants. Using this system, we have constructed mutants of Synechocystis 6803 that are modified in the region of the C-terminus of the D1 polypeptide. Characterization of these mutants has revealed that (1) processing of the D1 polypeptide is blocked when the residue after the cleavage site is changed from serine to proline (mutant Ser345Pro) with the result that the manganese cluster is unable to assemble correctly; (2) the C-terminal extension of 16 amino acid residues can be deleted with little consequence either for insertion of D1 into the thylakoid membrane or for assembly of D1 into a fully active PSII complex; (3) removal of only one more residue (mutant Ala344stop) results in a loss of assembly of the manganese cluster; and (4) the ability of detergent-solubilized PSII core complexes (lacking the manganese cluster) to bind and oxidize exogenous Mn2+ by the secondary donor, Z+, is largely unaffected in the processing mutants (the Ser345Pro mutant of Synechocystis 6803 and the LF-1 mutant of Scenedesmus obliquus) and the truncation mutant Ala344stop. Our results are consistent with a role for processing in regulating the assembly of the photosynthetic manganese cluster and a role for the free carboxy terminus of the mature D1 polypeptide in the ligation of one or more manganese ions of the cluster.     

Cytochrome c oxidase from bakers' yeast. III. Physical characterization of isolated subunits and chemical evidence for two different classes of polypeptides.

Earlier studies have shown that cytochrome c oxidase from bakers' yeast is an oligomeric enzyme which contains three polypeptides (I to III) synthesized on mitochondrial ribosomes and four polypeptides (IV to VII) synthesized on cytoplasmic ribosomes. These polypeptide subunits have now been isolated by a simple protocol which utilizes differences in polypeptide charge, solubility, and size. Their molecular weights determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, gel filtration in the presence of guanidine hydrochloride, and amino acid analysis were: I, 40,000; II, 33,000; III, 22,000; IV, 14,500; V, 12,700; VI, 12,700; and VII, 4,600. All seven polypeptide subunits exhibited acidic isoelectric points; cytoplasmically made subunits were more acidic than mitochondrially made ones. The amino acid composition of two mitochondrially made subunits and two cytoplasmically made subunits was determined. The two mitochondrial translation products, I and II, contained only 34.7% and 42.1% polar amino acids, respectively, whereas the two cytoplasmic translation products, IV and VI, contained 48.3% and 49.3%, respectively. This agreed with the observation that Subunits I and II are very insoluble, requiring detergents for solubility, whereas Subunits IV and VI are water-soluble in the absence of any added detergent. These results indicate that the cytochrome c oxidase subunits synthesized on mitochondrial and cytoplasmic ribosomes are fundamentally different in size, isoelectric properties, and hydrophobicity. They also suggest the possibility that at least some of the mitochondrially made subunits are buried in the lipid phase of the mitochondrial inner membrane.