Oligomycin Resistance Research Articles

Cryptococcus neoformans releases proteins during intracellular residence that affect the outcome of the fungal-macrophage interaction.

Cryptococcus neoformans is a facultative intracellular pathogen that can replicate and disseminate in mammalian macrophages. In this study, we analyzed fungal proteins identified in murine macrophage-like cells after infection with C. neoformans. To accomplish this, we developed a protocol to identify proteins released from cryptococcal cells inside macrophage-like cells; we identified 127 proteins of fungal origin in infected macrophage-like cells. Among the proteins identified was urease, a known virulence factor, and others such as transaldolase and phospholipase D, which have catalytic activities that could contribute to virulence. This method provides a straightforward methodology to study host–pathogen interactions. We chose to study further Yeast Oligomycin Resistance (Yor1), a relatively uncharacterized protein belonging to the large family of ATP binding cassette transporter (ABC transporters). These transporters belong to a large and ancient protein family found in all extant phyla. While ABC transporters have an enormous diversity of functions across varied species, in pathogenic fungi they are better studied as drug efflux pumps. Analysis of C. neoformans yor1Δ strains revealed defects in nonlytic exocytosis, capsule size, and dimensions of extracellular vesicles, when compared to wild-type strains. We detected no difference in growth rates and cell body size. Our results indicate that C. neoformans releases a large suite of proteins during macrophage infection, some of which can modulate fungal virulence and are likely to affect the fungal–macrophage interaction.

Bioinformatics analysis of genes of Streptomyces xinghaiensis (fradiae) ATCC 19609 with a focus on mutations conferring resistance to oligomycin A and its derivatives

ObjectivesThe aim of this study was to obtain Streptomyces xinghaiensis (fradiae) ATCC 19609 mutants resistant to oligomycin A and its derivatives and to identify the underlying mechanism of resistance. This study was based on the premise that S. xinghaiensis ATCC 19609 contains several oligomycin A biological targets, explaining why the strain remains supersensitive to oligomycin A despite all efforts to obtain resistant mutants using standard genetic methods. MethodsThe method to obtain oligomycin A-resistant mutants was performed in two steps: first, mutants slightly resistant to an oligomycin A derivative with an attenuated effect were obtained; and second, oligomycin A-resistant mutants were obtained from those mutants obtained earlier. The genomes of the mutants were then sequenced and a bioinformatics analysis of the detected mutations was conducted. ResultsMutants with seven mutations were required to obtain oligomycin A-resistant mutant strains of S. xinghaiensis characterised by a level of resistance comparable with that of the model organism Streptomyces lividans. Five of these mutations caused amino acid substitutions in the well-known oligomycin A biological target, namely the F0F1-ATP synthase A subunit, and the others caused amino acid substitutions in unexplored biological targets, including RecB-like recombinase, type IV helicase, DNA ligase and single-domain response regulator. ConclusionA new oligomycin resistance mechanism involving a pathway that repairs double-strand breaks in DNA known as non-homologous end joining (NHEJ) was discovered.

Substitution of Yor1p NBD1 residues improves the thermal stability of Human Cystic Fibrosis Transmembrane Conductance Regulator.

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a plasma membrane chloride channel protein that regulates vertebrate fluid homeostasis. The inefficiency of wild type human CFTR protein folding/trafficking is exacerbated by genetic mutations that can cause protein misfolding in the endoplasmic reticulum (ER) and subsequent degradation. This project investigates small changes in protein sequence that can alter the thermal stability of the large multi-domain CFTR protein. We target a conserved 70-residue α-subdomain located in the first nucleotide-binding domain that hosts the common misfolding mutation ∆F508. To investigate substitutions that can stabilize this domain, we constructed chimeras between human CFTR and its closest yeast homolog Yor1p. The α-subdomain of Yor1p was replaced with that of CFTR in Saccharomyces cerevisiae. Cellular localization of green fluorescence protein-tagged Yor1p-CFTR chimeras was analyzed by fluorescence microscopy and quantitative multispectral imaging flow cytometry, steady-state protein levels were compared by SDS-PAGE and protein function probed by a phenotypic oligomycin resistance assay. The chimeras exhibited ER retention in yeast characteristic of defective protein folding/processing. Substitution of seven CFTR α-subdomain residues that are highly conserved in Yor1p and other transporters but differ in CFTR (S495P/R516K/F533L/A534P/K536G/I539T/R553K) improved Yor1p-CFTR chimera localization to the yeast plasma membrane. When introduced into human CFTR expressed in mammalian cells, the same substitutions improve the purified protein thermal stability. This stabilized human CFTR protein will be directly useful for structural and biophysical studies that have been limited by the thermal sensitivity of wild type CFTR. The insights into critical structural residues within CFTR could facilitate development of effective therapeutics for CF-causing mutations.

Ribosomal Stalk Protein Silencing Partially Corrects the ΔF508-CFTR Functional Expression Defect.

The most common cystic fibrosis (CF) causing mutation, deletion of phenylalanine 508 (ΔF508 or Phe508del), results in functional expression defect of the CF transmembrane conductance regulator (CFTR) at the apical plasma membrane (PM) of secretory epithelia, which is attributed to the degradation of the misfolded channel at the endoplasmic reticulum (ER). Deletion of phenylalanine 670 (ΔF670) in the yeast oligomycin resistance 1 gene (YOR1, an ABC transporter) of Saccharomyces cerevisiae phenocopies the ΔF508-CFTR folding and trafficking defects. Genome-wide phenotypic (phenomic) analysis of the Yor1-ΔF670 biogenesis identified several modifier genes of mRNA processing and translation, which conferred oligomycin resistance to yeast. Silencing of orthologues of these candidate genes enhanced the ΔF508-CFTR functional expression at the apical PM in human CF bronchial epithelia. Although knockdown of RPL12, a component of the ribosomal stalk, attenuated the translational elongation rate, it increased the folding efficiency as well as the conformational stability of the ΔF508-CFTR, manifesting in 3-fold augmented PM density and function of the mutant. Combination of RPL12 knockdown with the corrector drug, VX-809 (lumacaftor) restored the mutant function to ~50% of the wild-type channel in primary CFTRΔF508/ΔF508 human bronchial epithelia. These results and the observation that silencing of other ribosomal stalk proteins partially rescue the loss-of-function phenotype of ΔF508-CFTR suggest that the ribosomal stalk modulates the folding efficiency of the mutant and is a potential therapeutic target for correction of the ΔF508-CFTR folding defect.

Atypical Subunit Composition of the Chlorophycean Mitochondrial F1FO-ATP Synthase and Role of Asa7 Protein in Stability and Oligomycin Resistance of the Enzyme

In yeast, mammals, and land plants, mitochondrial F(1)F(O)-ATP synthase (complex V) is a remarkable enzymatic machinery that comprises about 15 conserved subunits. Peculiar among eukaryotes, complex V from Chlamydomonadales algae (order of chlorophycean class) has an atypical subunit composition of its peripheral stator and dimerization module, with nine subunits of unknown evolutionary origin (Asa subunits). In vitro, this enzyme exhibits an increased stability of its dimeric form, and in vivo, Chlamydomonas reinhardtii cells are insensitive to oligomycins, which are potent inhibitors of proton translocation through the F(O) moiety. In this work, we showed that the atypical features of the Chlamydomonadales complex V enzyme are shared by the other chlorophycean orders. By biochemical and in silico analyses, we detected several atypical Asa subunits in Scenedesmus obliquus (Sphaeropleales) and Chlorococcum ellipsoideum (Chlorococcales). In contrast, complex V has a canonical subunit composition in other classes of Chlorophytes (Trebouxiophyceae, Prasinophyceae, and Ulvophyceae) as well as in Streptophytes (land plants), and in Rhodophytes (red algae). Growth, respiration, and ATP levels in Chlorophyceae were also barely affected by oligomycin concentrations that affect representatives of the other classes of Chlorophytes. We finally studied the function of the Asa7 atypical subunit by using RNA interference in C. reinhardtii. Although the loss of Asa7 subunit has no impact on cell bioenergetics or mitochondrial structures, it destabilizes in vitro the enzyme dimeric form and renders growth, respiration, and ATP level sensitive to oligomycins. Altogether, our results suggest that the loss of canonical components of the complex V stator happened at the root of chlorophycean lineage and was accompanied by the recruitment of novel polypeptides. Such a massive modification of complex V stator features might have conferred novel properties, including the stabilization of the enzyme dimeric form and the shielding of the proton channel. In these respects, we discuss an evolutionary scenario for F(1)F(O)-ATP synthase in the whole green lineage (i.e., Chlorophyta and Streptophyta).

F0F1 ATP synthase of Streptomycetes: Modulation of activity and oligomycin resistance by protein Ser/Thr kinases

Inverted membrane vesicles of Gram-positive actinobacteria Streptomyces fradiae, S. lividans, and S. avermitilis have been prepared and membrane-bound F0F1 ATP synthase has been biochemically characterized. It has been shown that the ATPase activity of membrane-bound F0F1 complex is Mg2+-dependent and moderately stimulated by high concentrations of Ca2+ ions (10–20 mM). The ATPase activity is inhibited by N,N′-dicyclohexylcarbodiimide and oligomycin A, typical F0F1 ATPase inhibitors that react with the membrane-bound F0 complex. The assay of biochemical properties of the F0F1 ATPases of Streptomycetes in all cases showed the presence of ATPase populations highly susceptible and insensitive to oligomycin A. The in vitro labeling and inhibitory assay showed that the inverted phospholipid vesicles of S. fradiae contained active membrane-bound Ser/Thr protein kinase(s) phosphorylating the proteins of the F0F1 complex. Inhibition of phosphorylation leads to decrease of the ATPase activity and increase of its susceptibility to oligomycin. The in vivo assay confirmed the enhancement of actinobacteria cell sensitivity to oligomycin after inhibition of endogenous phosphorylation. The sequencing of the S. fradiae genes encoding oligomycin-binding A and C subunits of F0F1 ATP synthase revealed their close phylogenetic relation to the genes of S. lividans and S. avermitilis.

Mapping of Interdomain Interfaces Required for the Functional Architecture of Yor1p, a Eukaryotic ATP-binding Cassette (ABC) Transporter

ATP-binding cassette (ABC) transporters are a large superfamily of proteins that mediate substrate translocation across biological membranes. Our goal was to define the intramolecular interactions that contribute to quaternary assembly of a eukaryotic ABC transporter and determine how the architecture of this protein influences its biogenesis within the secretory pathway. We used chemical cross-linking approaches to map interdomain interactions in the yeast ABC transporter, Yor1p, which functions as a pleiotropic drug pump at the plasma membrane. We have defined interactions between the two nucleotide-binding domains (NBDs) and between the NBDs and specific intracellular loops (ICLs) that are consistent with current structural models of bacterial ABC exporters. Furthermore, we detected relatively weak NBD-NBD and ICL-ICL interactions that may correspond to transient sites of cross-talk between domains required for coupling of ATP hydrolysis with substrate translocation. Mutation of a key residue in ICL2 caused misassembly of the altered protein, leading to increased sensitivity to the mitochondrial poison, oligomycin. We identified intragenic suppressing mutations that rescued the oligomycin resistance associated with this aberrant protein and demonstrated that the suppressing mutations restored multiple interdomain interfaces. Together, our biochemical and genetic approaches contribute to a greater understanding of the architecture of this important class of proteins and provide insight into the quality control surveillance that regulates their biogenesis and deployment within the eukaryotic cell.

Functional characterization of the Saccharomyces cerevisiae ABC-transporter Yor1p overexpressed in plasma membranes

Yor1p, a Saccharomyces cerevisiae plasma membrane ABC-transporter, is associated to oligomycin resistance and to rhodamine B transport. Here, by using the overexpressing strain Superyor [A. Decottignies, A.M. Grant, J.W. Nichols, H. de Wet, D.B. McIntosh, A. Goffeau, ATPase and multidrug transport activities of the overexpressed yeast ABC protein Yor1p, J. Biol. Chem. 273 (1998) 12612–12622], we show that Yor1p also confers resistance to rhodamine 6G and to doxorubicin. In addition, Yor1p protects cells, although weakly, against tetracycline, verapamil, eosin Y and ethidium bromide. The basal ATPase activity of the overexpressed form of Yor1p was studied in membrane preparations. This activity is quenched upon addition of micromolar amounts of vanadate. V max and K m values of ∼ 0.8 s − 1 and 50 ± 8 μM are measured. Mutations of essential residues in the nucleotide binding domain 2 reduces the activity to that measured with a Δ yor1 strain. ATP hydrolysis is strongly inhibited by the addition of potential substrates of the transporter. Covalent reaction of 8-azido-[α- 32P]ATP with Yor1p is not sensitive to the presence of excess oligomycin. Thus, competition of the drug with ATP binding is unlikely. Finally, we inspect possible hypotheses accounting for substrate inhibition, rather than stimulation, of ATP hydrolysis by the membrane preparation.

Stable Transformation of CHO Cells and Human NARP Cybrids Confers Oligomycin Resistance (olir) Following Transfer of a Mitochondrial DNA–Encoded olirATPase6 Gene to the Nuclear Genome: A Model System for mtDNA Gene Therapy

Point and deletion mutations and a general depletion of mammalian mitochondrial DNA (mtDNA) give rise to a wide variety of medical syndromes that are refractory to treatment, possibly including aging itself. While gene therapy directed at correcting such deficits in the mitochondrial genome may offer some therapeutic benefits, there are inherent problems associated with a direct approach. These problems are primarily due to the high mitochondrial genome copy number in each cell and the mitochondrial genome being "protected" inside the double-membrane mitochondrial organelle. In an alternative approach there is evidence that genes normally present in the mitochondrial genome can be incorporated into the nuclear genome. To extend such studies, we modified the Chinese Hamster Ovary (CHO) mtDNA-located ATPase6 gene (possessing a mutation which confers oligomycin resistance- oli(r)) by altering the mtDNA code to the universal code (U-code) to permit the correct translation of its mRNA in the cytoplasm. The U-code construct was inserted into the nuclear genome (nucDNA) of a wild type CHO cell. The expressed transgene products enabled the transformed CHO cell lines to grow in up to 1000 ng mL(-1) oligomycin, while untransformed sensitive CHO cells were eliminated in 1 ng mL(-1) oligomycin. This approach, termed allotopic expression, provides a model that may make possible the transfer of all 13 mtDNA mammalian protein-encoding genes to the nucDNA, for treatments of mtDNA disorders. The CHO mtATPase6 protein is 85% identical to both the mouse and human mtATPase6 protein; these proteins are highly conserved in the region of the oligomycin resistance mutation. They are also well conserved in the regions of the oligomycin resistance mutation of the mouse, and in the region of a mutation found in Leigh's syndrome (T8993G), also called NARP (neurogenic weakness, ataxia, retinitis pigmentosum). It is likely that the CHO oli(r) mtATPase6 Ucode construct could impart oligomycin-resistance in human and mouse cells, as well as function in place of the mutant ATPase subunit in a NARP cell line. Preliminary experiments on human cybrids homoplasmic for the NARP mutation (kindly supplied by D.C. Wallace), transformed with our construct, display an increased oligomycin resistance that supports these suppositions.

Identification of Interdependent Signals Required for Anterograde Traffic of the ATP-binding Cassette Transporter Protein Yor1p

The plasma membrane ATP-binding cassette (ABC) transporter Yor1p mediates oligomycin resistance in Saccharomyces cerevisiae. Its protein sequence places it in the multidrug resistance protein/cystic fibrosis transmembrane conductance regulator subfamily of ABC transporters. A key regulatory step in the biogenesis of this family of ABC transporter proteins is at the level of transport from the endoplasmic reticulum (ER) on through the secretory pathway. To explore the protein sequence requirements for Yor1p to move from the ER to its site of function at the plasma membrane, a series of truncation and alanine replacement mutations were constructed in Yor1p. This analysis detected two sequence motifs similar to the DXE element that has recently been found in other proteins that exit the ER. Loss of the N-terminal DXE element eliminated function of the protein, whereas loss of the C-terminal element only slightly reduced function of the resulting mutant Yor1p. Strikingly, although both of the single mutant proteins were stable, production of the double mutant caused dramatic destabilization of Yor1p. These data suggest that this large polytopic membrane protein requires multiple signals for normal forward trafficking, and elimination of this information may cause the mutant protein to be transferred to a degradative fate.

Cystic Fibrosis Transmembrane Regulator Regulates Uptake of Sphingoid Base Phosphates and Lysophosphatidic Acid: MODULATION OF CELLULAR ACTIVITY OF SPHINGOSINE 1-PHOSPHATE

Sphingolipids have been implicated in the regulation of cell growth, differentiation, and programmed cell death. Sphingosine 1-phosphate (SPP) has recently emerged as an important lipid messenger and a ligand for the endothelial differentiation gene receptor family of proteins through which it mediates its biologic effects. Recent studies in Saccharomyces cerevisiae in our laboratory implicated the yeast oligomycin resistance gene (YOR1), a member of the ATP binding cassette family of proteins, in the transport of SPP. The cystic fibrosis transmembrane regulator is a unique member of the ATP binding cassette transporter family and has high homology with YOR1. We therefore set out to investigate if this member of the family can regulate SPP transport. We demonstrate that C127/cystic fibrosis transmembrane regulator (CFTR) cells, expressing wild type CFTR, exhibited significantly higher uptake of sphingosine 1-phosphate than either cells expressing a mutant CFTR C127/DeltaF508 or C127/mock-transfected cells. This effect was specific, dose-dependent, and competed off by dihydrosphingosine 1-phosphate and lysophosphatidic acid. There was no difference in uptake of sphingosine, C(16)-ceramide, sphingomyelin, lysophingomyelin, phosphatidylcholine, lysophosphatidylcholine, or phosphatidic acid among the different cell lines. Pretreatment with forskolin or isobutylmethylxanthine to stimulate cAMP did not affect the uptake in any of the cell lines. Moreover, we found that mitogen-activated protein kinase activation by SPP was less responsive in C127/CFTR as compared with C127/mock-transfected cells, suggesting that uptake of SPP by CFTR may divert it from interacting with its cell surface receptors and attenuate signaling functions. Taken together, these data implicate CFTR in uptake of SPP and the related phosphorylated lipids dihydrosphingosine 1-phosphate and lysophosphatidic acid. This uptake influences the availability of SPP to modulate biologic activity via endothelial differentiation gene receptors. These studies may have important implications to cystic fibrosis.

Interaction between mitochondria derived from incompatible black Aspergillus isolates.

As black Aspergillus isolates are highly heterokaryon-incompatible mitochondrial transmissions were performed by protoplast fusion. Donor strains with oligomycin-resistant mitochondria and sensitive recipient partners of various A. japonicus isolates were applied and the progeny were selected for oligomycin resistance and for recipient nuclear phenotype. These strains basically inherited the mitochondrial DNA of the donor strain, which might remain unchanged (substituted progeny) or might be modified by specific sequences of the recipient mtDNAs (recombinant progeny). Different mobile elements characteristic of the recipient parents were exclusively responsible for the development of the feature of recombinant mtDNAs. Substituted progeny were either stable wild-type-like strains as a result of compatible co-operation between donor mitochondria and recipient nuclei, or aconidial strains with a reduced fitness, exhibiting a certain instability. The latter type was probably due to the less compatible communication between nuclear and extrachromosomal genetic systems originating from different parents. These progeny were able to undergo some developmental (segregation) processes during subsequent cultivation, resulting in a stable, wild-type phenotype which possessed a new type of mtDNA resembling that of the acceptor parents.

Cross-talk between Transcriptional Regulators of Multidrug Resistance in Saccharomyces cerevisiae

Multiple or pleiotropic drug resistance often arises in the yeast Saccharomyces cerevisiae due to genetic alterations of the functional state of the Cys(6)-Zn(II)(2) transcription factors Pdr1p and Pdr3p. Single amino acid substitutions give rise to hyperactive forms of these regulatory proteins, which in turn cause overproduction of downstream target genes that directly mediate multidrug resistance. Previous work has identified a novel Cys(6)-Zn(II)(2) transcription factor designated Yrr1p as mutant forms of this protein confer high level resistance to the cell cycle inhibitor reveromycin A and DNA damaging agent 4-nitroquinoline-N-oxide. In the present study, we demonstrate that Yrr1p also mediates oligomycin resistance through activation of the ATP-binding cassette transporter-encoding gene YOR1. Additionally, insertion of triplicated copies of the hemagglutinin epitope in the C-terminal region of Yrr1p causes the protein to behave as a hyperactive regulator of transcription. We have found that YRR1 expression is both controlled in a Pdr1p/Pdr3p-dependent manner and autoregulated. Chromatin immunoprecipitation experiments also show that Yrr1p associates with target promoters in vivo. Together these data argue that the signal generated by activation of Pdr1p and/or Pdr3p can be amplified through the action of these transcriptional regulatory proteins on downstream target genes, like YRR1, that also encode transcription factors.

Mutational disruption of plasma membrane trafficking of Saccharomyces cerevisiae Yor1p, a homologue of mammalian multidrug resistance protein.

The ATP binding cassette (ABC) transporter protein Yor1p was identified on the basis of its ability to elevate oligomycin resistance when it was overproduced from a high-copy-number plasmid. Analysis of the predicted amino acid sequence of Yor1p indicated that this protein was a new member of a subfamily of ABC transporter proteins defined by the multidrug resistance protein (MRP). In this work, Yor1p is demonstrated to localize to the Saccharomyces cerevisiae plasma membrane by both indirect immunofluorescence and biochemical fractionation studies. Several mutations were generated in the amino-terminal nucleotide binding domain (NBD1) of Yor1p to test if the high degree of sequence conservation in this region of the protein was important for function. Deletion of a phenylalanine residue at Yor1p position 670 led to a mutant protein that appeared to be retained in the endoplasmic reticulum (ER) and that was unstable. As shown by others, deletion of the analogous residue from a second mammalian MRP family member, the cystic fibrosis transmembrane conductance regulator (CFTR), also led to retention of this normally plasma membrane-localized protein in the ER. Changes in the spacing between or the sequences flanking functional motifs of Yor1p NBD1 led to defective trafficking or decreased activity of the mutant proteins. Analyses of the degradation of wild-type and DeltaF670 Yor1p indicated that the half-life of DeltaF670 Yor1p was dramatically shortened. While the vacuole was the primary site for turnover of wild-type Yor1p, degradation of DeltaF670 Yor1p was found to be more complex with both proteasomal and vacuolar contributions.

Intra- and Interspecies Virus Transfer in Aspergilli via Protoplast Fusion

Intra- and interspecies transfer of dsRNA viruses between blackAspergilliandAspergillus nidulansstrains has been investigated using protoplast fusion. We found interspecies transfer of virus in all combinations of blackAspergillusandA. nidulansstrains and vice versa. Using the same conditions, intraspecies virus transfer among heterokaryon incompatible strains was also tested. Whereas such transfer was always found amongA. nidulansstrains, transfer among blackAspergilliwas frequently unsuccessful. The lack of virus transfer between blackAspergillusisolates was further investigated by using a mitochondrial oligomycin resistance marker as a positive control for cytoplasmic exchange. These experiments showed independent transfer of the oligomycin resistance and dsRNA viruses during protoplast fusion of heterokaryon incompatible blackAspergilli. The inefficient transfer of dsRNA viruses between blackAspergilliis not caused by absolute resistance to viruses but may be related to heterokaryon incompatibility reactions that operate intraspecifically. Consequences for the dynamics of mycoviruses in populations of blackAspergilliare discussed.

Topology Mapping of the Amino-terminal Half of Multidrug Resistance-associated Protein by Epitope Insertion and Immunofluorescence

The multidrug resistance-associated protein (MRP) is an integral membrane protein that causes multidrug resistance when overexpressed in mammalian cells. Within the ATP-binding cassette superfamily, MRP belongs to a subgroup of structurally and functionally related proteins that includes the yeast cadmium factor 1 and yeast oligomycin resistance I proteins, and the mammalian sulfonylurea receptors SUR1 and SUR2. Hydropathy analysis of these proteins predicts a unique membrane-associated region at the amino terminus followed by a structural unit composed of 12 transmembrane (TM) domains and two nucleotide-binding domains that is characteristic of eukaryotic ATP-binding cassette transporters. The topology of the membrane-associated regions of MRP remains largely unknown and was investigated. Small hemagglutinin epitopes (YPYDVPDYAS) were inserted in predicted hydrophilic segments of the membrane-associated regions from the amino-terminal half of MRP and these proteins were expressed in HeLa cells, and tested for their capacity to confer etoposide resistance. The polarity of the inserted tags with respect to plasma membrane was then deduced by immunofluorescence in intact and permeabilized cells. Insertion of epitopes at positions 4, 163, 271, 574, and 653 produced functional proteins while insertions at positions 127, 417, 461, and 529 abrogated the capacity of MRP to confer drug resistance. Epitopes inserted at positions 4, 163, and 574 were localized extracellularly, whereas those inserted at positions 271 and 653 revealed an intracellular location. Although a single epitope inserted at position 461 was compatible with MRP function, it was inaccessible to the anti-epitope antibody and two copies of the tag at that site abrogated MRP function. These results indicate that the amino terminus of MRP is extracellular, while the linker segment joining the first and second membrane-associated regions is intracellular as is the first nucleotide-binding domain. Our findings are therefore consistent with a topological model of MRP that contains 5 TM segments in the first membrane-associated region and 6 TM segments in the second membrane region.

Recombination of mitochondrial DNAs following transmission of mitochondria among incompatible strains of black Aspergilli.

Successful intra- and interspecific mitochondrial transfers were performed by polyethylene glycol (PEG)-induced protoplast fusion among incompatible strains belonging to the Aspergillus niger species aggregate. The mitochondrial DNAs (mtDNAs) of the strains examined were of three main types based on their restriction fragment length polymorphism (RFLP) profiles. mtDNA types 1 and 2 correspond to A. niger and A. tubingensis species, respectively, while type 3 is represented by some Brazilian wild-type isolates (possibly a distinct species or subspecies). mtDNA types 1 and 2 could be further divided into several subgroups (1a-1e and 2a-2f). All these strains, representing different RFLP groups or subgroups, were fully incompatible with respect to nuclear complementation. The transfer experiments were carried out under selection pressure, using a mitochondrial oligomycin-resistant mutant of mtDNA type 1a as donor. Following fusion mitochondrial oligomycin-resistant progenies were recovered in the presence of oligomycin by selecting for the nuclear phenotypes of the oligomycin-sensitive recipient strains. All attempted transfers were successful, and resulted in different varieties of resistant recombinant mitochondrial progenies at various frequencies. Within the group of strains of mtDNA type 1, the transfer of oligomycin-resistant mitochondria resulted in the appearance of a single recombinant type of RFLP profile in each case. The recombination events were more complex when the transfer of oligomycin resistance occurred between strains representing different species (mtDNA groups 1a-->2 and 1a-->3). A great variety of recombinant mtDNA RFLP profiles appeared. Explanation for this phenomenon are discussed on the basis of preliminary physical mapping data.

Sequence analysis of a near-subtelomeric 35.4 kb DNA segment on the right arm of chromosome VII from Saccharomyces cerevisiae carrying the MAL1 locus reveals 15 complete open reading frames, including ZUO1, BGL2 and BIO2 genes and an ABC transporter gene.

The nucleotide sequence of 35,400 bp at approximately 10 kb from the right telomere of chromosome VII was determined. The segment contains the MAL1 locus, one of the five unlinked loci sufficient for maltose utilization. Until now, each of these loci was considered to contain three genes (for regulator, permease and alpha-glucosidase), but a fourth gene, presumably an extra alpha-glucosidase gene, was found at MAL1 adjacent to the usual cluster of three genes. The two glucosidase genes are present in opposite orientation, forming an inverted repeat structure. In addition to the four genes at MAL1, there are 11 complete, non-overlapping open reading frames (ORFs) longer than 300 bp in the sequence presented here. A new ABC transporter gene (YGR281w), required for oligomycin resistance was found (YOR1; Katzman et al., 1995), and the previously sequenced BGL2 (YGR282c), ZUO1 (YGR285c) and BIO2 (YGR286c) genes were located. The sequence of BIO2, a biotin synthetase gene, required substantial correction and the size of Bio2p is 375, rather than 356, amino acids. Two ORFs show rather weak similarities to animal genes: YGR278w to an unknown ORF of Caenorhabditis elegans and YGR284c to the murine Surf-4, a member of a cluster of at least four housekeeping genes. The remaining five ORFs do not encode known functions, but three of these show weak to high similarities to other ORFs in the Saccharomyces cerevisiae genome and one (YGR280c) codes for a particularly lysine-rich protein.

Mitochondrial inheritance in Aspergillus nidulans.

Mitochondrial chloramphenicol and oligomycin resistance mutations were used to investigate mitochondrial inheritance in A. nidulans. Mitochondrial RFLPs could not be used to distinguish between paternal and maternal mitochondria because none were detected in the 54 isolates investigated. Several thousand ascospores from each of 111 hybrid cleistothecia from 21 different crosses between 7 heterokaryon incompatible isolates were tested for biparental inheritance. All mitochondrial inheritance was strictly uniparental. Not one instance of paternal inheritance of mitochondria was observed. The implications of our results for the theory that uniparental inheritance evolved to avoid cytoplasmic conflict are discussed. Possible explanations for the maintenance of strict uniparental inheritance of mitochondria in an inbreeding homothallic organism are suggested. The chloramphenicol resistance marker was inherited preferentially to the oligomycin resistance marker probably due to the inhibited energy production of mitochondria with the oligomycin resistance mutation. The maternal parent was determined for 93 hybrid cleistothecia from 17 crosses between 7 different strains. Contrary to previous reports A. nidulans strains functioned as both maternal and paternal parent in most crosses.

Expression of an ATP-Binding Cassette Transporter-Encoding Gene (YOR1) Is Required for Oligomycin Resistance in Saccharomyces cerevisiae

Semidominant mutations in the PDR1 or PDR3 gene lead to elevated resistance to cycloheximide and oligomycin. PDR1 and PDR3 have been demonstrated to encode zinc cluster transcription factors. Cycloheximide resistance mediated by PDR1 and PDR3 requires the presence of the PDR5 membrane transporter-encoding gene. However, PDR5 is not required for oligomycin resistance. Here, we isolated a gene that is necessary for PDR1- and PDR3-mediated oligomycin resistance. This locus, designated YOR1, causes a dramatic elevation in oligomycin resistance when present in multiple copies. A yor1 strain exhibits oligomycin hypersensitivity relative to an isogenic wild-type strain. In addition, loss of the YOR1 gene blocks the elevation in oligomycin resistance normally conferred by mutant forms of PDR1 or PDR3. The YOR1 gene product is predicted to be a member of the ATP-binding cassette transporter family of membrane proteins. Computer alignment indicates that Yor1p shows striking sequence similarity with multidrug resistance-associated protein, Saccharomyces cerevisiae Ycf1p, and the cystic fibrosis transmembrane conductance regulator. Use of a YOR1-lacZ fusion gene indicates that YOR1 expression is responsive to PDR1 and PDR3. While PDR5 expression is strictly dependent on the presence of PDR1 or PDR3, control of YOR1 expression has a significant PDR1/PDR3-independent component. Taken together, these data indicate that YOR1 provides the link between transcriptional regulation by PDR1 and PDR3 and oligomycin resistance of yeast cells.