Functional Characterization of
            <i>Pneumocystis carinii brl1</i>
            by Transspecies Complementation Analysis

Presti, Libera Lo; Cockell, Moira; Cerutti, Lorenzo; Hauser, Philippe M.

doi:10.1128/ec.00321-07

Cited by 18 publications

(23 citation statements)

References 19 publications

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“…Members of the Brr6/Brl1 family of proteins are found in all fungi and lower eukaryotes that undergo closed mitosis. Whereas S. cerevisiae and many closely related fungi have both genes, S. pombe, Pneumocystis carinii, and other fungi distantly related to S. cerevisiae have a unique gene that is more closely related to Brl1 than to Brr6 (19,22). Brl1 is not a nucleoporin; its distribution in the NE is unaffected in strains carrying nucleoporin mutations that cause NPCs to cluster together (18,21).…”

mentioning

confidence: 99%

Yeast Integral Membrane Proteins Apq12, Brl1, and Brr6 Form a Complex Important for Regulation of Membrane Homeostasis and Nuclear Pore Complex Biogenesis

et al. 2015

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bProper functioning of intracellular membranes is critical for many cellular processes. A key feature of membranes is their ability to adapt to changes in environmental conditions by adjusting their composition so as to maintain constant biophysical properties, including fluidity and flexibility. Similar changes in the biophysical properties of membranes likely occur when intracellular processes, such as vesicle formation and fusion, require dramatic changes in membrane curvature. Similar modifications must also be made when nuclear pore complexes (NPCs) are constructed within the existing nuclear membrane, as occurs during interphase in all eukaryotes. Here we report on the role of the essential nuclear envelope/endoplasmic reticulum (NE/ER) protein Brl1 in regulating the membrane composition of the NE/ER. We show that Brl1 and two other proteins characterized previously-Brr6, which is closely related to Brl1, and Apq12-function together and are required for lipid homeostasis. All three transmembrane proteins are localized to the NE and can be coprecipitated. As has been shown for mutations affecting Brr6 and Apq12, mutations in Brl1 lead to defects in lipid metabolism, increased sensitivity to drugs that inhibit enzymes involved in lipid synthesis, and strong genetic interactions with mutations affecting lipid metabolism. Mutations affecting Brl1 or Brr6 or the absence of Apq12 leads to hyperfluid membranes, because mutant cells are hypersensitive to agents that increase membrane fluidity. We suggest that the defects in nuclear pore complex biogenesis and mRNA export seen in these mutants are consequences of defects in maintaining the biophysical properties of the NE. T he nuclear envelope (NE) of eukaryotic cells compartmentalizes the nuclear material and separates it from the cytoplasm. The double membrane of the NE consists of an outer and an inner nuclear membrane (ONM and INM) that differ in protein and lipid composition. The NE is structurally and functionally related to the endoplasmic reticulum (ER), and the ONM is contiguous with the ER (1, 2). Embedded in the NE are the nuclear pore complexes (NPCs) and, in budding yeast, the spindle pole body (SPB). NPCs are extremely large and are constructed from multiple copies of about 30 different nucleoporins (nups) (3, 4). NPCs mediate selective trafficking of proteins and other macromolecules between the nucleus and the cytoplasm but also serve other important functions, including gene activation and mRNA surveillance (5, 6). The biogenesis of NPCs and their distribution over the NE are highly regulated processes and are coordinated with the cell cycle (7). During interphase, the number of NPCs doubles. In budding yeast, the NE remains intact throughout the cell cycle, and all the formation of NPCs occurs through de novo construction within the NE.In addition to the ONM and the INM, the NE contains a pore membrane domain (POM), formed by the fusion of the INM and ONM at sites where NPCs are assembled (8). The POM is a highly curved region of the NE that is i...

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confidence: 99%

Yeast Integral Membrane Proteins Apq12, Brl1, and Brr6 Form a Complex Important for Regulation of Membrane Homeostasis and Nuclear Pore Complex Biogenesis

et al. 2015

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“…Assessment of the extracted DNAs was done by amplification of the unrelated S. cerevisiae BRL1 gene encoding an essential nuclear membrane protein (18). The primers used were 5=-GAA ACT CTT GGT ACA GAG G-3= and 5=-TGA TCT GTC CCA GTT GTG-3=.…”

Section: Methodsmentioning

confidence: 99%

“…The lack of a culture method complicates the identification of new drug targets in P. jirovecii. The strategy used so far has been to identify potential drug targets in the genome of Pneumocystis carinii, the species infecting rats, which was used as model (16)(17)(18)(19)(20). The existing antifungal agents and their targets in P. carinii have been reviewed (21).…”

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confidence: 99%

Functional Characterization of the Pneumocystis jirovecii Potential Drug Targets dhfs and abz2 Involved in Folate Biosynthesis

Luraschi

Cissé

Monod

et al. 2015

Antimicrob Agents Chemother

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cPneumocystis species are fungal parasites colonizing mammal lungs with strict host specificity. Pneumocystis jirovecii is the human-specific species and can turn into an opportunistic pathogen causing severe pneumonia in immunocompromised individuals. This disease is currently the second most frequent life-threatening invasive fungal infection worldwide. The most efficient drug, cotrimoxazole, presents serious side effects, and resistance to this drug is emerging. The search for new targets for the development of new drugs is thus of utmost importance. The recent release of the P. jirovecii genome sequence opens a new era for this task. It can now be carried out on the actual targets to be inhibited instead of on those of the relatively distant model Pneumocystis carinii, the species infecting rats. We focused on the folic acid biosynthesis pathway because (i) it is widely used for efficient therapeutic intervention, and (ii) it involves several enzymes that are essential for the pathogen and have no human counterparts. In this study, we report the identification of two such potential targets within the genome of P. jirovecii, the dihydrofolate synthase (dhfs) and the aminodeoxychorismate lyase (abz2). The function of these enzymes was demonstrated by the rescue of the null allele of the orthologous gene of Saccharomyces cerevisiae. P neumocystis organisms are extracellular fungi that colonize the lungs of mammals (1, 2). Each species displays strict host specificity for a given mammalian species. These fungi are thought to be obligate biotrophic parasites whose evolution has been marked by gene losses (3-7). Pneumocystis jirovecii is the human-specific species whose reservoir would be only humans (8). P. jirovecii can turn into an opportunistic pathogen that causes severe pneumonia in immunocompromised individuals (Pneumocystis jirovecii pneumonia [PCP]). This disease is currently the second most frequent life-threatening invasive fungal infection worldwide, with Ͼ400,000 cases annually (9).The current drug of choice for prophylaxis and treatment of PCP is cotrimoxazole, a combination of sulfamethoxazole and trimethoprim. The two latter drugs are inhibitors of dihydropteroate synthase (DHPS) and dihydrofolate reductase (DHFR), respectively. These two enzymes are involved in the biosynthesis of folic acid, a metabolite that is required for the biosynthesis of crucial cellular components. Organisms such as Pneumocystis and other lower eukaryotes can synthesize their own folic acid, whereas this compound is a vitamin obtained from food for mammals. Experiments in the rat animal model suggested that the antiPneumocystis activity of cotrimoxazole might be mainly due to the sulfamethoxazole (10). The widespread use of cotrimoxazole for the prevention of PCP since 1989 has been correlated with an increase in the prevalence of specific mutations within the putative active site of DHPS, similar to those observed in other pathogens resistant to cotrimoxazole. These mutations were associated with a breakthrough of prophy...

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“…Transformations and complementation assays in fission and budding yeasts were performed as described previously (36).…”

Section: Methodsmentioning

confidence: 99%

“…Identification of such modules in the genome of the rat-specific pathogen P. carinii may help to identify novel targets for directed pharmaceutical intervention to treat opportunistic infection of immunocompromised human hosts by P. jirovecii. To identify conserved functional modules, we are assessing the ability of genes from P. carinii to function in the budding yeast Saccharomyces cerevisiae as well as in the fission yeast Schizosaccharomyces pombe, the closest evolutionary relationship for which molecular genetic analysis is well developed (25,36).…”

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confidence: 99%

Functional Differentiation of tbf1 Orthologues in Fission and Budding Yeasts

et al. 2009

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In Saccharomyces cerevisiae, TBF1, an essential gene, influences telomere function but also has other roles in the global regulation of transcription. We have identified a new member of the tbf1 gene family in the mammalian pathogen Pneumocystis carinii. We demonstrate by transspecies complementation that its ectopic expression can provide the essential functions of Schizosaccharomyces pombe tbf1 but that there is no rescue between fission and budding yeast orthologues. Our findings indicate that an essential function of this family of proteins has diverged in the budding and fission yeasts and suggest that effects on telomere length or structure are not the primary cause of inviability in S. pombe tbf1 null strains.The phylogenetic pedigree of Pneumocystis species implies they are likely to employ conserved fungus-specific proteins in pathways that are essential for viability. Identification of such modules in the genome of the rat-specific pathogen P. carinii may help to identify novel targets for directed pharmaceutical intervention to treat opportunistic infection of immunocompromised human hosts by P. jirovecii. To identify conserved functional modules, we are assessing the ability of genes from P. carinii to function in the budding yeast Saccharomyces cerevisiae as well as in the fission yeast Schizosaccharomyces pombe, the closest evolutionary relationship for which molecular genetic analysis is well developed (25,36).In the course of this study, we identified a potential homologue of the S. pombe tbf1 (Sptbf1) and S. cerevisiae TBF1 (ScTBF1) genes. Sequence comparisons indicate that both SpTbf1p and ScTbf1p are members of a conserved fungusspecific family (50, 51). All the Tbf1 family members contain a C-terminal "telobox" DNA binding domain (8, 9) but bear additional significant homology throughout their coding sequences. The telobox, a particular variant of the Myb family motif, is also found in the mammalian telomere factors TRF1 and TRF2 as well as in the fission yeast telomeric protein Taz1p (13). The telobox recognizes sequences similar to the mammalian telomeric repeat TTAGGG. High-affinity binding sites for ScTbf1p have also been identified in the STARs (subtelomeric antisilencing regions) of the subtelomeric X and YЈ elements. Several studies have proposed a regulatory role for ScTbf1p at telomeres (2,6,19,33). The ScTBF1 gene is essential, but this is commonly assumed to be due to its function as a global transcriptional regulator that binds to many sites throughout chromatin rather than to direct effects on telomeres (10, 33).SpTbf1p was identified in S. pombe whole-cell extracts as one of several activities that exhibited differential affinity in vitro for tandem copies of the human and S. pombe telomere repeat sequences (55, 62). The Sptbf1 gene is essential, although its full range of cellular functions is unknown. Overexpression of Sptbf1 has been shown to slightly increase the mean length of telomeres in vivo (51).We have examined whether the P. carinii tbf1 homologue (Pctbf1) can rescue ...

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Functional Characterization of Pneumocystis carinii brl1 by Transspecies Complementation Analysis

Cited by 18 publications

References 19 publications

Yeast Integral Membrane Proteins Apq12, Brl1, and Brr6 Form a Complex Important for Regulation of Membrane Homeostasis and Nuclear Pore Complex Biogenesis

Yeast Integral Membrane Proteins Apq12, Brl1, and Brr6 Form a Complex Important for Regulation of Membrane Homeostasis and Nuclear Pore Complex Biogenesis

Functional Characterization of the Pneumocystis jirovecii Potential Drug Targets dhfs and abz2 Involved in Folate Biosynthesis

Functional Differentiation of tbf1 Orthologues in Fission and Budding Yeasts

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