A negative genetic interaction map in isogenic cancer cell lines reveals cancer cell vulnerabilities

Improved efforts are necessary to define the functional product of cancer mutations currently being revealed through large‐scale sequencing efforts. Using genome‐scale pooled shRNA screening technology, we mapped negative genetic interactions across a set of isogenic cancer cell lines and confirmed hundreds of these interactions in orthogonal co‐culture competition assays to generate a high‐confidence genetic interaction network of differentially essential or differential essentiality (DiE) genes. The network uncovered examples of conserved genetic interactions, densely connected functional modules derived from comparative genomics with model systems data, functions for uncharacterized genes in the human genome and targetable vulnerabilities. Finally, we demonstrate a general applicability of DiE gene signatures in determining genetic dependencies of other non‐isogenic cancer cell lines. For example, the PTEN^?/? DiE genes reveal a signature that can preferentially classify PTEN‐dependent genotypes across a series of non‐isogenic cell lines derived from the breast, pancreas and ovarian cancers. Our reference network suggests that many cancer vulnerabilities remain to be discovered through systematic derivation of a network of differentially essential genes in an isogenic cancer cell model.     

Tracking Glideosome-associated protein 50 reveals the development and organization of the inner membrane complex of Plasmodium falciparum

The most deadly of the human malaria parasites, Plasmodium falciparum, has different stages specialized for invasion of hepatocytes, erythrocytes, and the mosquito gut wall. In each case, host cell invasion is powered by an actin-myosin motor complex that is linked to an inner membrane complex (IMC) via a membrane anchor called the glideosome-associated protein 50 (PfGAP50). We generated P. falciparum transfectants expressing green fluorescent protein (GFP) chimeras of PfGAP50 (PfGAP50-GFP). Using immunoprecipitation and fluorescence photobleaching, we show that C-terminally tagged PfGAP50-GFP can form a complex with endogenous copies of the linker protein PfGAP45 and the myosin A tail domain-interacting protein (MTIP). Full-length PfGAP50-GFP is located in the endoplasmic reticulum in early-stage parasites and then redistributes to apical caps during the formation of daughter merozoites. In the final stage of schizogony, the PfGAP50-GFP profile extends further around the merozoite surface. Three-dimensional (3D) structured illumination microscopy reveals the early-stage IMC as a doubly punctured flat ellipsoid that separates to form claw-shaped apposed structures. A GFP fusion of PfGAP50 lacking the C-terminal membrane anchor is misdirected to the parasitophorous vacuole. Replacement of the acid phosphatase homology domain of PfGAP50 with GFP appears to allow correct trafficking of the chimera but confers a growth disadvantage.     

A maxi-chloride channel in the inner membrane of mammalian mitochondria

Patch-clamp experiments on swollen mitochondria of human, mouse and rat origins have revealed activity by an approximately 400 pS (in 150 mM KCl), voltage-dependent and anion-selective channel. This channel is located in the inner membrane, as shown by experiments with mitochondria from cells expressing a fluorescent mitochondrial tag protein and by the co-presence of the 107 pS channel and of the permeability transition pore (PTP). The frequency of appearance was inversely related to the presence of the PTP. This and the comparison of its electrophysiological characteristics with those of the PTP indicate that it is closely related to the latter, possibly corresponding to a monomeric unit whose dimer constitutes the full PTP. The channel is similar but not identical to isolated-and-reconstituted mitochondrial porin, and it is present also in mitochondria from cells lacking porin isoforms. Its identification with porin is therefore to be excluded. It most likely coincides instead with the “maxi-chloride channel” characterized in the plasma membrane of various cell types.     

A maxi-chloride channel in the inner membrane of mammalian mitochondria

Patch-clamp experiments on swollen mitochondria of human, mouse and rat origins have revealed activity by an approximately 400 pS (in 150 mM KCl), voltage-dependent and anion-selective channel. This channel is located in the inner membrane, as shown by experiments with mitochondria from cells expressing a fluorescent mitochondrial tag protein and by the co-presence of the 107 pS channel and of the permeability transition pore (PTP). The frequency of appearance was inversely related to the presence of the PTP. This and the comparison of its electrophysiological characteristics with those of the PTP indicate that it is closely related to the latter, possibly corresponding to a monomeric unit whose dimer constitutes the full PTP. The channel is similar but not identical to isolated-and-reconstituted mitochondrial porin, and it is present also in mitochondria from cells lacking porin isoforms. Its identification with porin is therefore to be excluded. It most likely coincides instead with the "maxi-chloride channel" characterized in the plasma membrane of various cell types.     

Charting the genetic interaction map of a cell

Genome sequencing projects have revealed a massive catalog of genes and astounding genetic diversity in a variety of organisms. We are now faced with the formidable challenge of assigning functions to thousands of genes, and how to use this information to understand how genes interact and coordinate cell function. Studies indicate that the majority of eukaryotic genes are dispensable, highlighting the extensive buffering of genomes against genetic and environmental perturbations. Such robustness poses a significant challenge to those seeking to understand the wiring diagram of the cell. Genome-scale screens for genetic interactions are an effective means to chart the network that underlies this functional redundancy. A complete atlas of genetic interactions offers the potential to assign functions to most genes identified by whole genome sequencing projects and to delineate a functional wiring diagram of the cell. Perhaps more importantly, mapping genetic networks on a large-scale will shed light on the general principles and rules governing genetic networks and provide valuable information regarding the important but elusive relationship between genotype and phenotype.     

A genetic map in the Mimulus guttatus species complex reveals transmission ratio distortion due to heterospecific interactions.

As part of a study of the genetics of floral adaptation and speciation in the Mimulus guttatus species complex, we constructed a genetic linkage map of an interspecific cross between M. guttatus and M. nasutus. We genotyped an F(2) mapping population (N = 526) at 255 AFLP, microsatellite, and gene-based markers and derived a framework map through repeated rounds of ordering and marker elimination. The final framework map consists of 174 marker loci on 14 linkage groups with a total map length of 1780 cM Kosambi. Genome length estimates (2011-2096 cM) indicate that this map provides thorough coverage of the hybrid genome, an important consideration for QTL mapping. Nearly half of the markers in the full data set (49%) and on the framework map (48%) exhibited significant transmission ratio distortion (alpha = 0.05). We localized a minimum of 11 transmission ratio distorting loci (TRDLs) throughout the genome, 9 of which generate an excess of M. guttatus alleles and a deficit of M. nasutus alleles. This pattern indicates that the transmission ratio distortion results from particular interactions between the heterospecific genomes and suggests that substantial genetic divergence has occurred between these Mimulus species. We discuss possible causes of the unequal representation of parental genomes in the F(2) generation.     

Both ATP and an energized inner membrane are required to import a purified precursor protein into mitochondria.

We have investigated the energy requirement of mitochondrial protein import with a simplified system containing only isolated yeast mitochondria, energy sources and a purified precursor protein. This precursor was a fusion protein composed of 22 residues of the cytochrome oxidase subunit IV pre-sequence fused to mouse dihydrofolate reductase. Import of this protein required not only an energized inner membrane, but also ATP. ATP could be replaced by GTP, but not by CTP, TTP or non-hydrolyzable ATP analogs. Added ATP did not increase the membrane potential of respiring mitochondria; it supported import even if the proton-translocating mitochondrial ATPase and the entry of ATP into the matrix were blocked. We conclude that ATP exerts its effect on mitochondrial protein import outside the inner membrane.     

Cyclosporin A is a potent inhibitor of the inner membrane permeability transition in liver mitochondria

The immunosuppressive peptide cyclosporin A is a powerful inhibitor of the Ca2+-dependent permeability transition in rat liver mitochondria. When swelling is used to monitor the transition, the inhibitor is effective regardless of whether N-ethylmaleimide, Hg2+, WY-14643, t-butyl hydroperoxide, oxalacetate, rhein, phosphate, phosphoenolpyruvate, or ruthenium red plus uncoupler is used as the inducing agent. Twenty-five to fifty pmol/mg protein of cyclosporin A reduces the swelling response by 50% with complete inhibition obtained at about 150 pmol/mg protein. The compound, which does not inhibit Ca2+ uptake or mitochondrial phospholipase A2, is effective when added before or after the transition promoting agent. These findings, together with the shape of the inhibition dose-response curve, suggest that cyclosporin A essentially titrates a mitochondrial component which is present at 80-90 pmol/mg protein. It is proposed that this component is a solute unselective, regulated pore or a factor involved in controlling such a structure.     

Substrate specificity of inner membrane peptidase in yeast mitochondria

The inner membrane protease (IMP) cleaves intra-organelle sorting peptides from precursor proteins in mitochondria of the yeast Saccharomyces cerevisiae. An unusual feature of the IMP is the presence of two catalytic subunits, Imp1p and Imp2p, which recognize distinct substrate sets even though both enzymes belong to the same protease family. This nonoverlapping substrate specificity was hypothesized to result from the recognition of distinct residues at the P′₁ position (also termed +1 position) in the protease substrates. Here, we constructed an extensive series of mutations to obtain a profile of the critical cleavage site residues in IMP substrates and conclude that Imp1p, and not Imp2p, recognizes specific P′₁ residues. In addition to its specificity for P′₁ residues, Imp1p also shows substrate specificity for the P₃ (−3) position. In contrast, Imp2p recognizes the P₁ (−1) position and the P₃ position. Based on this new understanding of IMP substrate specificity, we conducted a survey for candidate IMP substrates in mammalian mitochondria and found consensus Imp2p cleavage sites in mammalian precursors to cytochrome c₁ and glycerol-3-phosphate (G-3-P) dehydrogenase. Presence of a putative Imp2p cleavage site in G-3-P dehydrogenase was surprising, as its yeast ortholog contains an Imp1p cleavage site. To address this issue experimentally, we performed the first co-expression of mammalian IMP with proposed mammalian IMP precursors in yeast and show that murine precursors to cytochrome c₁ and G-3-P dehydrogenase are cleaved by murine Imp2p. These results suggest, surprisingly, G-3-P dehydrogenase has switched from Imp1p in yeast to Imp2p in mammals.     

Evidence for the existence of an inner membrane anion channel in mitochondria

Mitochondria normally exhibit very low electrophoretic permeabilities to physiologically important anions such as chloride, bicarbonate, phosphate, succinate, citrate, etc. Nevertheless, considerable evidence has accumulated which suggests that heart and liver mitochondria contain a specific anion-conducting channel. In this review, a postulated inner membrane anion channel is discussed in the context of other known pathways for anion transport in mitochondria. This anion channel exhibits the following properties. It is anion-selective and inhibited physiologically by protons and magnesium ions. It is inhibited reversibly by quinine and irreversibly by dicyclohexylcarbodiimide. We propose that the inner membrane anion channel is formed by inner membrane proteins and that this pathway is normally latent due to regulation by matrix Mg2+. The physiological role of the anion channel is unknown; however, this pathway is well designed to enable mitochondria to restore their normal volume following pathological swelling. In addition, the inner membrane anion channel provides a potential futile cycle for regulated non-shivering thermogenesis and may be important in controlled energy dissipation.     

A novel complex of membrane proteins required for formation of a spherical nucleus.

Two membrane proteins were identified through their genetic interaction with the nucleoporin Nup84p and shown to participate in nuclear envelope morphogenesis in yeast. One component is a known sporulation factor Spo7p, and the other, Nem1p, a novel protein whose C-terminal domain is conserved during eukaryotic evolution. Spo7p and Nem1p localize to the nuclear/ER membrane and behave biochemically as integral membrane proteins. Nem1p binds to Spo7p via its conserved C-terminal domain. Although cells without Spo7p or Nem1p are viable, they exhibit a drastically altered nuclear morphology with long, pore-containing double nuclear membrane extensions. These protrusions emanate from a core nucleus which contains the DNA, and penetrate deeply into the cytoplasm. Interestingly, not only Spo7(-) and Nem1(-), but also several nucleoporin mutants are defective in sporulation. Thus, Spo7p and Nem1p, which exhibit a strong genetic link to nucleoporins of the Nup84p complex, fulfil an essential role in formation of a spherical nucleus and meiotic division.     

A linkage map reveals a complex basis for segregation distortion in an interpopulation cross in the moss Ceratodon purpureus

We report the construction of a linkage map for the moss Ceratodon purpureus (n = 13), based on a cross between geographically distant populations, and provide the first experimental confirmation of maternal chloroplast inheritance in bryophytes. From a mapping population of 288 recombinant haploid gametophytes, genotyped at 121 polymorphic AFLP loci, three gene-based nuclear loci, one chloroplast marker, and sex, we resolved 15 linkage groups resulting in a map length of approximately 730 cM. We estimate that the map covers more than three-quarters of the C. purpureus genome. Approximately 35% of the loci were sex linked, not including those in recombining pseudoautosomal regions. Nearly 45% of the loci exhibited significant segregation distortion (alpha = 0.05). Several pairs of unlinked distorted loci showed significant deviations from multiplicative genotypic frequencies, suggesting that distortion arises from genetic interactions among loci. The distorted autosomal loci all exhibited an excess of the maternal allele, suggesting that these interactions may involve nuclear-cytoplasmic factors. The sex ratio of the progeny was significantly male biased, and the pattern of nonrandom associations among loci indicates that this results from interactions between the sex chromosomes. These results suggest that even in interpopulation crosses, multiple mechanisms act to influence segregation ratios.     

ER membrane protein complex required for nuclear fusion

Diploid cells of the yeast Saccharomyces cerevisiae form after the mating of two haploid cells of the opposite mating type. After fusion of the two plasma membranes of the mating cells, a dinucleated cell forms initially in which the two haploid nuclei then rapidly fuse to form a single diploid nucleus. This latter event, called karyogamy, can be divided into two distinct steps: the microtubule-based movement that causes the two nuclei to become closely juxtaposed and the fusion of the nuclear membranes. For the membrane fusion step, one required component, the ER luminal protein Kar2p (BiP), has been identified. For topological reasons, however, it has been unclear how Kar2p could function in this role. Kar2p is localized to the luminal (i.e., noncytoplasmic) face of the ER membrane, yet nuclear fusion must initiate from the cytosolic side of the outer nuclear membrane or the ER membrane with which it is contiguous. There is both genetic and biochemical evidence that Kar2p interacts with Sec63p, an ER membrane protein containing both luminal and cytosolic domains that is involved in protein translocation across the membrane. We have isolated novel sec63 mutant alleles that display severe karyogamy defects. Disruption of the genes encoding other Sec63p-associated proteins (Sec71p and Sec72p) also results in karyogamy defects. A suppressor mutant (sos1-1) partially corrects the translocation defect but does not alleviate the karyogamy defect. sec61 and sec62 mutant alleles that cause similar or more severe protein translocation defects show no karyogamy defects. Taken together, these results suggest a direct role for Sec63p, Sec71p, and Sec72p in nuclear membrane fusion and argue against the alternative interpretation that the karyogamy defects result as an indirect consequence of the impaired membrane translocation of another component(s) required for the process. We propose that an ER/nuclear membrane protein complex composed of Sec63p, Sec71p, and Sec72p plays a central role in mediating nuclear membrane fusion and requires ER luminally associated Kar2p for its function.     

Properties of the inner membrane anion channel in intact mitochondria

The mitochondrial inner membrane possesses an anion channel (IMAC) which mediates the electrophoretic transport of a wide variety of anions and is believed to be an important component of the volume homeostatic mechanism. IMAC is regulated by matrix Mg²⁺ (IC₅₀=38 µM at pH 7.4) and by matrix H⁺ (pIC₅₀=7.7). Moreover, inhibition by Mg²⁺ is pH-dependent. IMAC is also reversibly inhibited by many cationic amphiphilic drugs, including propranolol, and irreversibly inhibited byN,N-dicyclohexylcarbodiimide. Mercurials have two effects on its activity: (1) they increase the IC₅₀ values for Mg²⁺, H⁺, and propranolol, and (2) they inhibit transport. The most potent inhibitor of IMAC is tributyltin, which blocks anion uniport in liver mitochondria at about 1 nmol/mg. The inhibitory dose is increased by mercurials; however, this effect appears to be unrelated to the other mercurial effects. IMAC also appears to be present in plant mitochondria; however, it is insensitive to inhibition by Mg²⁺, mercurials, andN,N-dicyclohexylcarbodiimide. Some inhibitors of the adenine nucleotide translocase also inhibit IMAC, including Cibacron Blue, agaric acid, and palmitoyl CoA; however, atractyloside has no effect.     

URF13, a ligand-gated,pore-forming receptor for T-toxin in the inner membrane ofcms-T mitochondria

URF13 is the product of a mitochondrial-encoded gene (T-urfl3) found only in maize plants containing the Texas male-sterile cytoplasm (cms-T), and it is thought to be responsible for both cytoplasmic male sterility and the susceptibility ofcms-T maize to the fungal pathogensBipolaris maydis race T andPhyllosticta maydis. Mitochondria isolated fromcms-T maize are uniquely sensitive to pathotoxins (T-toxin) produced by these fungi and to methomyl (a commercial insecticide). URF13 acts as a receptor that specifically binds T-toxin to produce hydrophilic pores in the inner mitochondrial membrane. When expressed inEscherichia coli cells, URF13 also forms hydrophilic pores in the plasma membrane if exposed to T-toxin or methomyl. Topological studies established that URF13 contains three membrane-spanning -helices, two of which are amphipathic and can contribute to pore formation. Chemical crosslinking of URF13 was used to demonstrate the existence of URF13 oligomers incms-T mitochondria andE. coli cells. The ability of the carboxylate-specific reagent,N,N-dicyclohexycarbodiimide, to cross-link URF13 was used in conjunction with site-directed mutagenesis to establish that the URF13 tetramer has a central core consisting of a four--helical bundle which undergoes a conformational change after interaction with T-toxin or methomyl. Overall, the experimental evidence indicates that URF13 functions as a ligand-gated, pore-forming T-toxin receptor incms-T mitochondria.     

Respiratory complex III is required to maintain complex I in mammalian mitochondria

A puzzling observation in patients with oxidative phosphorylation (OXPHOS) deficiencies is the presence of combined enzyme complex defects associated with a genetic alteration in only one protein-coding gene. In particular, mutations in the mtDNA encoded cytochrome b gene are associated either with combined complex I+III deficiency or with only complex III deficiency. We have reproduced the combined complex I+III defect in mouse and human cultured cell models harboring cytochrome b mutations. In both, complex III assembly is impeded and causes a severe reduction in the amount of complex I, not observed when complex III activity was pharmacologically inhibited. Metabolic labeling in mouse cells revealed that complex I was assembled, although its stability was severely hampered. Conversely, complex III stability was not influenced by the absence of complex I. This structural dependence among complexes I and III was confirmed in a muscle biopsy of a patient harboring a nonsense cytochrome b mutation.     

Supramolecular organization of protein complexes in the mitochondrial inner membrane

The liquid state model that envisions respiratory chain complexes diffusing freely in the membrane is increasingly challenged by reports of supramolecular organization of the complexes in the mitochondrial inner membrane. Supercomplexes of complex III with complex I and/or IV can be isolated after solubilisation with mild detergents like digitonin. Electron microscopic studies have shown that these have a distinct architecture and are not random aggregates. A 3D reconstruction of a I1III2IV1 supercomplex shows that the ubiquinone and cytochrome c binding sites of the individual complexes are facing each other, suggesting a role in substrate channelling. Formation of supercomplexes plays a role in the assembly and stability of the complexes, suggesting that the supercomplexes are the functional state of the respiratory chain. Furthermore, a supramolecular organisation of ATP synthases has been observed in mitochondria, where ATP synthase is organised in dimer rows. Dimers can be isolated by mild detergent extraction and recent electron microscopic studies have shown that the membrane domains of the two partners in the dimer are at an angle to each other, indicating that in vivo the dimers would cause the membrane to bend. The suggested role in crista formation is supported by the observation of rows of ATP synthase dimers in the most curved parts of the cristae. Together these observations show that the mitochondrial inner membrane is highly organised and that the molecular events leading to ATP synthesis are carefully coordinated.     

Periplasmic protein-protein contacts in the inner membrane protein Wzc form a tetrameric complex required for the assembly of Escherichia coli group 1 capsules

The K antigenic capsular polysaccharide forms a structural layer, the capsule, on the surfaces of Escherichia coli cells. The capsule provides an important protective covering that helps protect encapsulated bacteria from host immune defenses. The assembly and translocation of the capsule requires proteins in the inner and outer membranes. The inner membrane protein Wzc is a tyrosine autokinase that plays an essential role in what is believed to be a coordinated biosynthesis and secretion process. Mutants lacking Wzc can form K antigen oligosaccharides but are unable to polymerize high molecular weight capsular polymers. Wzc homologs have been identified in exopolymer biosynthesis systems in many different Gram-negative and -positive bacteria. Using single particle averaging on cryo-negatively stained samples, we have produced the first three-dimensional structure of this type of membrane protein in its phosphorylated state at approximately 14 A resolution. Perfluoro-octanoate-PAGE analysis of detergent-solubilized oligomeric Wzc and symmetry analysis of the transmission electron microscopy data clearly demonstrated that Wzc forms a tetrameric complex with C4 rotational symmetry. Viewed from the top of the complex, the oligomer is square with a diameter of approximately 100 A and can be divided into four separate densities. From the side, Wzc is approximately 110 A high and has a distinctive appearance similar to an extracted molar tooth. The upper "crown" region is approximately 55 A high and forms a continuous ring of density. Four unconnected "roots" ( approximately 65 A high) emerge from the underside of the crown. We propose that the crown is formed by protein-protein contacts from the four Wzc periplasmic domains, while each root represents an individual cytoplasmic tyrosine autokinase domain.     

Identification of a multicomponent complex required for outer membrane biogenesis in Escherichia coli

Gram-negative bacteria have an outer membrane (OM) that functions as a barrier to protect the cell from toxic compounds such as antibiotics and detergents. The OM is a highly asymmetric bilayer composed of phospholipids, glycolipids, and proteins. Assembly of this essential organelle occurs outside the cytoplasm in an environment that lacks obvious energy sources such as ATP, and the mechanisms involved are poorly understood. We describe the identification of a multiprotein complex required for the assembly of proteins in the OM of Escherichia coli. We also demonstrate genetic interactions between genes encoding components of this protein assembly complex and imp, which encodes a protein involved in the assembly of lipopolysaccharides (LPS) in the OM. These genetic interactions suggest a role for YfgL, one of the lipoprotein components of the protein assembly complex, in a homeostatic control mechanism that coordinates the overall OM assembly process.