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1.  Evolution of Chemical Diversity in Echinocandin Lipopeptide Antifungal Metabolites 
Eukaryotic Cell  2015;14(7):698-718.
The echinocandins are a class of antifungal drugs that includes caspofungin, micafungin, and anidulafungin. Gene clusters encoding most of the structural complexity of the echinocandins provided a framework for hypotheses about the evolutionary history and chemical logic of echinocandin biosynthesis. Gene orthologs among echinocandin-producing fungi were identified. Pathway genes, including the nonribosomal peptide synthetases (NRPSs), were analyzed phylogenetically to address the hypothesis that these pathways represent descent from a common ancestor. The clusters share cooperative gene contents and linkages among the different strains. Individual pathway genes analyzed in the context of similar genes formed unique echinocandin-exclusive phylogenetic lineages. The echinocandin NRPSs, along with the NRPS from the inp gene cluster in Aspergillus nidulans and its orthologs, comprise a novel lineage among fungal NRPSs. NRPS adenylation domains from different species exhibited a one-to-one correspondence between modules and amino acid specificity that is consistent with models of tandem duplication and subfunctionalization. Pathway gene trees and Ascomycota phylogenies are congruent and consistent with the hypothesis that the echinocandin gene clusters have a common origin. The disjunct Eurotiomycete-Leotiomycete distribution appears to be consistent with a scenario of vertical descent accompanied by incomplete lineage sorting and loss of the clusters from most lineages of the Ascomycota. We present evidence for a single evolutionary origin of the echinocandin family of gene clusters and a progression of structural diversification in two fungal classes that diverged approximately 290 to 390 million years ago. Lineage-specific gene cluster evolution driven by selection of new chemotypes contributed to diversification of the molecular functionalities.
PMCID: PMC4486670  PMID: 26024901
2.  Unraveling the Function of the Response Regulator BcSkn7 in the Stress Signaling Network of Botrytis cinerea 
Eukaryotic Cell  2015;14(7):636-651.
Important for the lifestyle and survival of every organism is the ability to respond to changing environmental conditions. The necrotrophic plant pathogen Botrytis cinerea triggers an oxidative burst in the course of plant infection and therefore needs efficient signal transduction to cope with this stress. The factors involved in this process and their precise roles are still not well known. Here, we show that the transcription factor Bap1 and the response regulator (RR) B. cinerea Skn7 (BcSkn7) are two key players in the oxidative stress response (OSR) of B. cinerea; both have a major influence on the regulation of classical OSR genes. A yeast-one-hybrid (Y1H) approach proved direct binding to the promoters of gsh1 and grx1 by Bap1 and of glr1 by BcSkn7. While the function of Bap1 is restricted to the regulation of oxidative stress, analyses of Δbcskn7 mutants revealed functions beyond the OSR. Involvement of BcSkn7 in development and virulence could be demonstrated, indicated by reduced vegetative growth, impaired formation of reproductive structures, and reduced infection cushion-mediated penetration of the host by the mutants. Furthermore, Δbcskn7 mutants were highly sensitive to oxidative, osmotic, and cell wall stress. Analyses of Δbap1 bcskn7 double mutants indicated that loss of BcSkn7 uncovers an underlying phenotype of Bap1. In contrast to Saccharomyces cerevisiae, the ortholog of the glutathione peroxidase Gpx3p is not required for nuclear translocation of Bap1. The presented results contribute to the understanding of the OSR in B. cinerea and prove that it differs substantially from that of yeast, demonstrating the complexity and versatility of components involved in signaling pathways.
PMCID: PMC4486671  PMID: 25934690
3.  Editorial Board 
Eukaryotic Cell  2015;14(7):A1.
PMCID: PMC4486672
4.  Articles of Significant Interest Selected from This Issue by the Editors 
Eukaryotic Cell  2015;14(7):625.
PMCID: PMC4486674
5.  The NDR Kinase Cbk1 Downregulates the Transcriptional Repressor Nrg1 through the mRNA-Binding Protein Ssd1 in Candida albicans 
Eukaryotic Cell  2015;14(7):671-683.
NDR (nuclear Dbf2-related) kinases are essential components for polarized morphogenesis, cytokinesis, cell proliferation, and apoptosis. The NDR kinase Cbk1 is required for the hyphal growth of Candida albicans; however, the molecular functions of Cbk1 in hyphal morphogenesis are largely unknown. Here, we report that Cbk1 downregulates the transcriptional repressor Nrg1 through the mRNA-binding protein Ssd1, which has nine Cbk1 phosphorylation consensus motifs. We found that deletion of SSD1 partially suppressed the defective hyphal growth of the C. albicans cbk1Δ/Δ mutant and that Ssd1 physically interacts with Cbk1. Cbk1 was required for Ssd1 localization to polarized growth sites. The phosphomimetic SSD1 allele (ssd1-9E) allowed the cbk1Δ/Δ mutant to form short hyphae, and the phosphodeficient SSD1 allele (ssd1-9A) resulted in shorter hyphae than did the wild-type SSD1 allele, indicating that Ssd1 phosphorylation by Cbk1 is important for hyphal morphogenesis. Furthermore, we show that the transcriptional repressor Nrg1 does not disappear during hyphal initiation in the cbk1Δ/Δ mutant but is completely absent in the cbk1Δ/Δ ssd1Δ/Δ double mutant. Deletion of SSD1 also increased Als3 expression and internalization of the cbk1Δ/Δ mutant in the human embryonic kidney cell line HEK293T. Collectively, our results suggest that one of the functions of Cbk1 in the hyphal morphogenesis of C. albicans is to downregulate Nrg1 through Ssd1.
PMCID: PMC4486675  PMID: 26002720
6.  Impact of Protein Palmitoylation on the Virulence Potential of Cryptococcus neoformans 
Eukaryotic Cell  2015;14(7):626-635.
The localization and specialized function of Ras-like proteins are largely determined by posttranslational processing events. In a highly regulated process, palmitoyl groups may be added to C-terminal cysteine residues, targeting these proteins to specific membranes. In the human fungal pathogen Cryptococcus neoformans, Ras1 protein palmitoylation is essential for growth at high temperature but is dispensable for sexual differentiation. Ras1 palmitoylation is also required for localization of this protein on the plasma membrane. Together, these results support a model in which specific Ras functions are mediated from different subcellular locations. We therefore hypothesize that proteins that activate Ras1 or mediate Ras1 localization to the plasma membrane will be important for C. neoformans pathogenesis. To further characterize the Ras1 signaling cascade mediating high-temperature growth, we have identified a family of protein S-acyltransferases (PATs), enzymes that mediate palmitoylation, in the C. neoformans genome database. Deletion strains for each candidate gene were generated by homogenous recombination, and each mutant strain was assessed for Ras1-mediated phenotypes, including high-temperature growth, morphogenesis, and sexual development. We found that full Ras1 palmitoylation and function required one particular PAT, Pfa4, and deletion of the PFA4 gene in C. neoformans resulted in altered Ras1 localization to membranes, impaired growth at 37°C, and reduced virulence.
PMCID: PMC4486677  PMID: 25862155
7.  Rgc2 Regulator of Glycerol Channel Fps1 Functions as a Homo- and Heterodimer with Rgc1 
Eukaryotic Cell  2015;14(7):719-725.
The plasma membrane aquaglyceroporin Fps1 is responsible for glycerol transport in yeast in response to changes in extracellular osmolarity. Fps1 functions as a homotetramer, and control of its channel activity in response to hyperosmotic shock involves a redundant pair of fungus-specific regulators, Rgc1 and Rgc2 (regulators of the glycerol channel), and the mitogen-activatd protein kinase (MAPK) Hog1 (high-osmolarity glycerol response). Rgc1 and Rgc2 maintain Fps1 in an open-channel state by binding to its C-terminal cytoplasmic domain. Phosphorylation of Rgc1 and Rgc2 by Hog1 induces their eviction from Fps1 and consequent channel closure. In the absence of Fps1 channel function, cells experience chronic cell wall stress, which may be exploited for antifungal drug development. We show here that Rgc1 and Rgc2 form homodimers and heterodimers with each other and that dimer formation of Rgc2 is mediated by its N-terminal domain. Mutations that prevent Rgc2 dimerization block its ability to open Fps1. Therefore, the Rgc-Rgc dimer interface might be an attractive drug target.
PMCID: PMC4486678  PMID: 26024902
8.  Biochemical and Kinetic Characterization of the Eukaryotic Phosphotransacetylase Class IIa Enzyme from Phytophthora ramorum 
Eukaryotic Cell  2015;14(7):652-660.
Phosphotransacetylase (Pta), a key enzyme in bacterial metabolism, catalyzes the reversible transfer of an acetyl group from acetyl phosphate to coenzyme A (CoA) to produce acetyl-CoA and Pi. Two classes of Pta have been identified based on the absence (PtaI) or presence (PtaII) of an N-terminal regulatory domain. PtaI has been fairly well studied in bacteria and one genus of archaea; however, only the Escherichia coli and Salmonella enterica PtaII enzymes have been biochemically characterized, and they are allosterically regulated. Here, we describe the first biochemical and kinetic characterization of a eukaryotic Pta from the oomycete Phytophthora ramorum. The two Ptas from P. ramorum, designated PrPtaII1 and PrPtaII2, both belong to class II. PrPtaII1 displayed positive cooperativity for both acetyl phosphate and CoA and is allosterically regulated. We compared the effects of different metabolites on PrPtaII1 and the S. enterica PtaII and found that, although the N-terminal regulatory domains share only 19% identity, both enzymes are inhibited by ATP, NADP, NADH, phosphoenolpyruvate (PEP), and pyruvate in the acetyl-CoA/Pi-forming direction but are differentially regulated by AMP. Phylogenetic analysis of bacterial, archaeal, and eukaryotic sequences identified four subtypes of PtaII based on the presence or absence of the P-loop and DRTGG subdomains within the N-terminal regulatory domain. Although the E. coli, S. enterica, and P. ramorum enzymes all belong to the IIa subclass, our kinetic analysis has indicated that enzymes within a subclass can still display differences in their allosteric regulation.
PMCID: PMC4486679  PMID: 25956919
9.  The AngFus3 Mitogen-Activated Protein Kinase Controls Hyphal Differentiation and Secondary Metabolism in Aspergillus niger 
Eukaryotic Cell  2015;14(6):602-615.
Adaptation to a changing environment is essential for the survival and propagation of sessile organisms, such as plants or fungi. Filamentous fungi commonly respond to a worsening of their growth conditions by differentiation of asexually or sexually produced spores. The formation of these specialized cell types is, however, also triggered as part of the general life cycle by hyphal age or density. Spores typically serve for dispersal and, therefore, translocation but can also act as resting states to endure times of scarcity. Eukaryotic differentiation in response to environmental and self-derived signals is commonly mediated by three-tiered mitogen-activated protein (MAP) kinase signaling cascades. Here, we report that the MAP kinase Fus3 of the black mold Aspergillus niger (AngFus3) and its upstream kinase AngSte7 control vegetative spore formation and secondary metabolism. Mutants lacking these kinases are defective in conidium induction in response to hyphal density but are fully competent in starvation-induced sporulation, indicating that conidiation in A. niger is triggered by various independent signals. In addition, the mutants exhibit an altered profile of volatile metabolites and secrete dark pigments into the growth medium, suggesting a dysregulation of the secondary metabolism. By assigning the AngFus3 MAP kinase pathway to the transduction of a potentially self-derived trigger, this work contributes to the unraveling of the intricate signaling networks controlling fungal differentiation. Moreover, our data further support earlier observations that differentiation and secondary metabolism are tightly linked in filamentous fungi.
PMCID: PMC4452566  PMID: 25888553
10.  Developmental Growth Control Exerted via the Protein A Kinase Tpk2 in Ashbya gossypii 
Eukaryotic Cell  2015;14(6):593-601.
Sporulation in Ashbya gossypii is induced by nutrient-limited conditions and leads to the formation of haploid spores. Using RNA-seq, we have determined a gene set induced upon sporulation, which bears considerable overlap with that of Saccharomyces cerevisiae but also contains A. gossypii-specific genes. Addition of cyclic AMP (cAMP) to nutrient-limited media blocks sporulation and represses the induction of sporulation specific genes. Deletion of the protein kinase A (PKA) catalytic subunits encoded by TPK1 and TPK2 showed reduced growth in tpk1 but enhanced growth in the tpk2 strain; however, both mutants sporulated well. Sporulation can be blocked by cAMP in tpk1 but not in tpk2 strains. Similarly, TPK2 acts at a second developmental switch promoting the break in spore dormancy. In S. cerevisiae, PKA phosphorylates and inhibits Msn2/4. The transcript profiles of the tpk1 and msn2/4 mutants were very similar to that of the wild type under sporulation conditions. However, deletion of the single A. gossypii MSN2/4 homolog generated a specific sporulation defect. We identified a set of genes involved in spore wall assembly that was downregulated in the msn2/4 mutant, particularly DIT2, suggesting that poor spore viability may be due to lysis of spores. Our results reveal specific functional differences between the two catalytic PKA subunits in A. gossypii and identified Tpk2 as the key A kinase that transduces developmental decisions of growth. Our data also suggest that Msn2/4 is involved only at a late step of sporulation in A. gossypii and is not a major regulator of IME1.
PMCID: PMC4452567  PMID: 25862153
11.  Overlapping Functions between SWR1 Deletion and H3K56 Acetylation in Candida albicans 
Eukaryotic Cell  2015;14(6):578-587.
Nucleosome destabilization by histone variants and modifications has been implicated in the epigenetic regulation of gene expression, with the histone variant H2A.Z and acetylation of H3K56 (H3K56ac) being two examples. Here we find that deletion of SWR1, the major subunit of the SWR1 complex depositing H2A.Z into chromatin in exchange for H2A, promotes epigenetic white-opaque switching in Candida albicans. We demonstrate through nucleosome mapping that SWR1 is required for proper nucleosome positioning on the promoter of WOR1, the master regulator of switching, and that its effects differ in white and opaque cells. Furthermore, we find that H2A.Z is enriched adjacent to nucleosome-free regions at the WOR1 promoter in white cells, suggesting a role in the stabilization of a repressive chromatin state. Deletion of YNG2, a subunit of the NuA4 H4 histone acetyltransferase (HAT) that targets SWR1 activity through histone acetylation, produces a switching phenotype similar to that of swr1, and both may act downstream of the GlcNAc signaling pathway. We further uncovered a genetic interaction between swr1 and elevated H3K56ac with the discovery that the swr1 deletion mutant is highly sensitive to nicotinamide. Our results suggest that the interaction of H2A.Z and H3K56ac regulates epigenetic switching at the nucleosome level, as well as having global effects.
PMCID: PMC4452568  PMID: 25862154
12.  Trypanosoma brucei Bloodstream Forms Depend upon Uptake of myo-Inositol for Golgi Complex Phosphatidylinositol Synthesis and Normal Cell Growth 
Eukaryotic Cell  2015;14(6):616-624.
myo-Inositol is a building block for all inositol-containing phospholipids in eukaryotes. It can be synthesized de novo from glucose-6-phosphate in the cytosol and endoplasmic reticulum. Alternatively, it can be taken up from the environment via Na+- or H+-linked myo-inositol transporters. While Na+-coupled myo-inositol transporters are found exclusively in the plasma membrane, H+-linked myo-inositol transporters are detected in intracellular organelles. In Trypanosoma brucei, the causative agent of human African sleeping sickness, myo-inositol metabolism is compartmentalized. De novo-synthesized myo-inositol is used for glycosylphosphatidylinositol production in the endoplasmic reticulum, whereas the myo-inositol taken up from the environment is used for bulk phosphatidylinositol synthesis in the Golgi complex. We now provide evidence that the Golgi complex-localized T. brucei H+-linked myo-inositol transporter (TbHMIT) is essential in bloodstream-form T. brucei. Downregulation of TbHMIT expression by RNA interference blocked phosphatidylinositol production and inhibited growth of parasites in culture. Characterization of the transporter in a heterologous expression system demonstrated a remarkable selectivity of TbHMIT for myo-inositol. It tolerates only a single modification on the inositol ring, such as the removal of a hydroxyl group or the inversion of stereochemistry at a single hydroxyl group relative to myo-inositol.
PMCID: PMC4452569  PMID: 25888554
13.  A Glycosylation Mutant of Trypanosoma brucei Links Social Motility Defects In Vitro to Impaired Colonization of Tsetse Flies In Vivo 
Eukaryotic Cell  2015;14(6):588-592.
Transmission of African trypanosomes by tsetse flies requires that the parasites migrate out of the midgut lumen and colonize the ectoperitrophic space. Early procyclic culture forms correspond to trypanosomes in the lumen; on agarose plates they exhibit social motility, migrating en masse as radial projections from an inoculation site. We show that an Rft1−/− mutant needs to reach a greater threshold number before migration begins, and that it forms fewer projections than its wild-type parent. The mutant is also up to 4 times less efficient at establishing midgut infections. Ectopic expression of Rft1 rescues social motility defects and restores the ability to colonize the fly. These results are consistent with social motility reflecting movement to the ectoperitrophic space, implicate N-glycans in the signaling cascades for migration in vivo and in vitro, and provide the first evidence that parasite-parasite interactions determine the success of transmission by the insect host.
PMCID: PMC4452570  PMID: 25862152
14.  Aspergillus nidulans Ambient pH Signaling Does Not Require Endocytosis 
Eukaryotic Cell  2015;14(6):545-553.
Aspergillus nidulans (Pal) ambient pH signaling takes place in cortical structures containing components of the ESCRT pathway, which are hijacked by the alkaline pH-activated, ubiquitin-modified version of the arrestin-like protein PalF and taken to the plasma membrane. There, ESCRTs scaffold the assembly of dedicated Pal proteins acting downstream. The molecular details of this pathway, which results in the two-step proteolytic processing of the transcription factor PacC, have received considerable attention due to the key role that it plays in fungal pathogenicity. While current evidence strongly indicates that the pH signaling role of ESCRT complexes is limited to plasma membrane-associated structures where PacC proteolysis would take place, the localization of the PalB protease, which almost certainly catalyzes the first and only pH-regulated proteolytic step, had not been investigated. In view of ESCRT participation, this formally leaves open the possibility that PalB activation requires endocytic internalization. As endocytosis is essential for hyphal growth, nonlethal endocytic mutations are predicted to cause an incomplete block. We used a SynA internalization assay to measure the extent to which any given mutation prevents endocytosis. We show that none of the tested mutations impairing endocytosis to different degrees, including slaB1, conditionally causing a complete block, have any effect on the activation of the pathway. We further show that PalB, like PalA and PalC, localizes to cortical structures in an alkaline pH-dependent manner. Therefore, signaling through the Pal pathway does not involve endocytosis.
PMCID: PMC4452571  PMID: 25841020
15.  Cryptococcus Strains with Different Pathogenic Potentials Have Diverse Protein Secretomes 
Eukaryotic Cell  2015;14(6):554-563.
Secreted proteins are the frontline between the host and pathogen. In mammalian hosts, secreted proteins enable invasive infection and can modulate the host immune response. Cryptococcosis, caused by pathogenic Cryptococcus species, begins when inhaled infectious propagules establish to produce pulmonary infection, which, if not resolved, can disseminate to the central nervous system to cause meningoencephalitis. Strains of Cryptococcus species differ in their capacity to cause disease, and the mechanisms underlying this are not well understood. To investigate the role of secreted proteins in disease, we determined the secretome for three genome strains of Cryptococcus species, including a hypovirulent and a hypervirulent strain of C. gattii and a virulent strain of C. neoformans. Sixty-seven unique proteins were identified, with different numbers and types of proteins secreted by each strain. The secretomes of the virulent strains were largely limited to proteolytic and hydrolytic enzymes, while the hypovirulent strain had a diverse secretome, including non-conventionally secreted canonical cytosolic and immunogenic proteins that have been implicated in virulence. The hypovirulent strain cannot establish pulmonary infection in a mouse model, but strains of this genotype have caused human meningitis. To directly test brain infection, we used intracranial inoculation and found that the hypovirulent strain was substantially more invasive than its hypervirulent counterpart. We suggest that immunogenic proteins secreted by this strain invoke a host response that limits pulmonary infection but that there can be invasive growth and damage if infection reaches the brain. Given their known role in virulence, it is possible that non-conventionally secreted proteins mediate this process.
PMCID: PMC4452572  PMID: 25841021
16.  Eaf1 Links the NuA4 Histone Acetyltransferase Complex to Htz1 Incorporation and Regulation of Purine Biosynthesis 
Eukaryotic Cell  2015;14(6):535-544.
Proper modulation of promoter chromatin architecture is crucial for gene regulation in order to precisely and efficiently orchestrate various cellular activities. Previous studies have identified the stimulatory effect of the histone-modifying complex NuA4 on the incorporation of the histone variant H2A.Z (Htz1) at the PHO5 promoter (A. Auger, L. Galarneau, M. Altaf, A. Nourani, Y. Doyon, R. T. Utley, D. Cronier, S. Allard, and J. Côté, Mol Cell Biol 28:2257–2270, 2008, In vitro studies with a reconstituted system also indicated an intriguing cross talk between NuA4 and the H2A.Z-loading complex, SWR-C (M. Altaf, A. Auger, J. Monnet-Saksouk, J. Brodeur, S. Piquet, M. Cramet, N. Bouchard, N. Lacoste, R. T. Utley, L. Gaudreau, J. Côté, J Biol Chem 285:15966–15977, 2010, In this work, we investigated the role of the NuA4 scaffold subunit Eaf1 in global gene expression and genome-wide incorporation of Htz1. We found that loss of Eaf1 affects Htz1 levels mostly at the promoters that are normally highly enriched in the histone variant. Analysis of eaf1 mutant cells by expression array unveiled a relationship between NuA4 and the gene network implicated in the purine biosynthesis pathway, as EAF1 deletion cripples induction of several ADE genes. NuA4 directly interacts with Bas1 activation domain, a key transcription factor of adenine genes. Chromatin immunoprecipitation (ChIP) experiments demonstrate that nucleosomes on the inactive ADE17 promoter are acetylated already by NuA4 and enriched in Htz1. Upon derepression, these poised nucleosomes respond rapidly to activate ADE gene expression in a mechanism likely reminiscent of the PHO5 promoter, leading to nucleosome disassembly. These detailed molecular events depict a specific case of cross talk between NuA4-dependent acetylation and incorporation of histone variant Htz1, presetting the chromatin structure over ADE promoters for subsequent chromatin remodeling and activated transcription.
PMCID: PMC4452573  PMID: 25841019
17.  Editorial Board 
Eukaryotic Cell  2015;14(6):A1.
PMCID: PMC4452574
18.  In Vivo Function of PTEX88 in Malaria Parasite Sequestration and Virulence 
Eukaryotic Cell  2015;14(6):528-534.
Malaria pathology is linked to remodeling of red blood cells by eukaryotic Plasmodium parasites. Central to host cell refurbishment is the trafficking of parasite-encoded virulence factors through the Plasmodium translocon of exported proteins (PTEX). Much of our understanding of its function is based on experimental work with cultured Plasmodium falciparum, yet direct consequences of PTEX impairment during an infection remain poorly defined. Using the murine malaria model parasite Plasmodium berghei, it is shown here that efficient sequestration to the pulmonary, adipose, and brain tissue vasculature is dependent on the PTEX components thioredoxin 2 (TRX2) and PTEX88. While TRX2-deficient parasites remain virulent, PTEX88-deficient parasites no longer sequester in the brain, correlating with abolishment of cerebral complications in infected mice. However, an apparent trade-off for virulence attenuation was spleen enlargement, which correlates with a strongly reduced schizont-to-ring-stage transition. Strikingly, general protein export is unaffected in PTEX88-deficient mutants that mature normally in vitro. Thus, PTEX88 is pivotal for tissue sequestration in vivo, parasite virulence, and preventing exacerbation of spleen pathology, but these functions do not correlate with general protein export to the host erythrocyte. The presented data suggest that the protein export machinery of Plasmodium parasites and their underlying mechanistic features are considerably more complex than previously anticipated and indicate challenges for targeted intervention strategies.
PMCID: PMC4452575  PMID: 25820521
19.  Articles of Significant Interest Selected from This Issue by the Editors 
Eukaryotic Cell  2015;14(6):527.
PMCID: PMC4452576
20.  Diversification of Paralogous α-Isopropylmalate Synthases by Modulation of Feedback Control and Hetero-Oligomerization in Saccharomyces cerevisiae 
Eukaryotic Cell  2015;14(6):564-577.
Production of α-isopropylmalate (α-IPM) is critical for leucine biosynthesis and for the global control of metabolism. The budding yeast Saccharomyces cerevisiae has two paralogous genes, LEU4 and LEU9, that encode α-IPM synthase (α-IPMS) isozymes. Little is known about the biochemical differences between these two α-IPMS isoenzymes. Here, we show that the Leu4 homodimer is a leucine-sensitive isoform, while the Leu9 homodimer is resistant to such feedback inhibition. The leu4Δ mutant, which expresses only the feedback-resistant Leu9 homodimer, grows slowly with either glucose or ethanol and accumulates elevated pools of leucine; this phenotype is alleviated by the addition of leucine. Transformation of the leu4Δ mutant with a centromeric plasmid carrying LEU4 restored the wild-type phenotype. Bimolecular fluorescent complementation analysis showed that Leu4-Leu9 heterodimeric isozymes are formed in vivo. Purification and kinetic analysis showed that the hetero-oligomeric isozyme has a distinct leucine sensitivity behavior. Determination of α-IPMS activity in ethanol-grown cultures showed that α-IPM biosynthesis and growth under these respiratory conditions depend on the feedback-sensitive Leu4 homodimer. We conclude that retention and further diversification of two yeast α-IPMSs have resulted in a specific regulatory system that controls the leucine–α-IPM biosynthetic pathway by selective feedback sensitivity of homomeric and heterodimeric isoforms.
PMCID: PMC4452578  PMID: 25841022
21.  N-Terminal Presequence-Independent Import of Phosphofructokinase into Hydrogenosomes of Trichomonas vaginalis 
Eukaryotic Cell  2015;14(12):1264-1275.
Mitochondrial evolution entailed the origin of protein import machinery that allows nuclear-encoded proteins to be targeted to the organelle, as well as the origin of cleavable N-terminal targeting sequences (NTS) that allow efficient sorting and import of matrix proteins. In hydrogenosomes and mitosomes, reduced forms of mitochondria with reduced proteomes, NTS-independent targeting of matrix proteins is known. Here, we studied the cellular localization of two glycolytic enzymes in the anaerobic pathogen Trichomonas vaginalis: PPi-dependent phosphofructokinase (TvPPi-PFK), which is the main glycolytic PFK activity of the protist, and ATP-dependent PFK (TvATP-PFK), the function of which is less clear. TvPPi-PFK was detected predominantly in the cytosol, as expected, while all four TvATP-PFK paralogues were imported into T. vaginalis hydrogenosomes, although none of them possesses an NTS. The heterologous expression of TvATP-PFK in Saccharomyces cerevisiae revealed an intrinsic capability of the protein to be recognized and imported into yeast mitochondria, whereas yeast ATP-PFK resides in the cytosol. TvATP-PFK consists of only a catalytic domain, similarly to “short” bacterial enzymes, while ScATP-PFK includes an N-terminal extension, a catalytic domain, and a C-terminal regulatory domain. Expression of the catalytic domain of ScATP-PFK and short Escherichia coli ATP-PFK in T. vaginalis resulted in their partial delivery to hydrogenosomes. These results indicate that TvATP-PFK and the homologous ATP-PFKs possess internal structural targeting information that is recognized by the hydrogenosomal import machinery. From an evolutionary perspective, the predisposition of ancient ATP-PFK to be recognized and imported into hydrogenosomes might be a relict from the early phases of organelle evolution.
PMCID: PMC4664869  PMID: 26475173
22.  Comparison of Switching and Biofilm Formation between MTL-Homozygous Strains of Candida albicans and Candida dubliniensis 
Eukaryotic Cell  2015;14(12):1186-1202.
Candida albicans and Candida dubliniensis are highly related species that share the same main developmental programs. In C. albicans, it has been demonstrated that the biofilms formed by strains heterozygous and homozygous at the mating type locus (MTL) differ functionally, but studies rarely identify the MTL configuration. This becomes a particular problem in studies of C. dubliniensis, given that one-third of natural strains are MTL homozygous. For that reason, we have analyzed MTL-homozygous strains of C. dubliniensis for their capacity to switch from white to opaque, the stability of the opaque phenotype, CO2 induction of switching, pheromone induction of adhesion, the effects of minority opaque cells on biofilm thickness and dry weight, and biofilm architecture in comparison with C. albicans. Our results reveal that C. dubliniensis strains switch to opaque at lower average frequencies, exhibit a far lower level of opaque phase stability, are not stimulated to switch by high CO2, exhibit more variability in biofilm architecture, and most notably, form mature biofilms composed predominately of pseudohyphae rather than true hyphae. Therefore, while several traits of MTL-homozygous strains of C. dubliniensis appear to be degenerating or have been lost, others, most notably several related to biofilm formation, have been conserved. Within this context, the possibility is considered that C. dubliniensis is transitioning from a hypha-dominated to a pseudohypha-dominated biofilm and that aspects of C. dubliniensis colonization may provide insights into the selective pressures that are involved.
PMCID: PMC4664870  PMID: 26432632
23.  Yeast Integral Membrane Proteins Apq12, Brl1, and Brr6 Form a Complex Important for Regulation of Membrane Homeostasis and Nuclear Pore Complex Biogenesis 
Eukaryotic Cell  2015;14(12):1217-1227.
Proper 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.
PMCID: PMC4664871  PMID: 26432634
24.  The ABCs of Candida albicans Multidrug Transporter Cdr1 
Eukaryotic Cell  2015;14(12):1154-1164.
In the light of multidrug resistance (MDR) among pathogenic microbes and cancer cells, membrane transporters have gained profound clinical significance. Chemotherapeutic failure, by far, has been attributed mainly to the robust and diverse array of these proteins, which are omnipresent in every stratum of the living world. Candida albicans, one of the major fungal pathogens affecting immunocompromised patients, also develops MDR during the course of chemotherapy. The pivotal membrane transporters that C. albicans has exploited as one of the strategies to develop MDR belongs to either the ATP binding cassette (ABC) or the major facilitator superfamily (MFS) class of proteins. The ABC transporter Candida drug resistance 1 protein (Cdr1p) is a major player among these transporters that enables the pathogen to outplay the battery of antifungals encountered by it. The promiscuous Cdr1 protein fulfills the quintessential need of a model to study molecular mechanisms of multidrug transporter regulation and structure-function analyses of asymmetric ABC transporters. In this review, we cover the highlights of two decades of research on Cdr1p that has provided a platform to study its structure-function relationships and regulatory circuitry for a better understanding of MDR not only in yeast but also in other organisms.
PMCID: PMC4664872  PMID: 26407965
25.  The New Shape of EC 
Eukaryotic Cell  2015;14(12):1151-1152.
The journal Eukaryotic Cell has served the eukaryotic microbiology community since 2002. It will continue to do so as it merges into the new broad-scope open-access journal mSphere in 2016.
PMCID: PMC4664875  PMID: 26622047

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