Flexibility of a Eukaryotic Lipidome – Insights from Yeast Lipidomics

Mass spectrometry-based shotgun lipidomics has enabled the quantitative and comprehensive assessment of cellular lipid compositions. The yeast Saccharomyces cerevisiae has proven to be a particularly valuable experimental system for studying lipid-related cellular processes. Here, by applying our shotgun lipidomics platform, we investigated the influence of a variety of commonly used growth conditions on the yeast lipidome, including glycerophospholipids, triglycerides, ergosterol as well as complex sphingolipids. This extensive dataset allowed for a quantitative description of the intrinsic flexibility of a eukaryotic lipidome, thereby providing new insights into the adjustments of lipid biosynthetic pathways. In addition, we established a baseline for future lipidomic experiments in yeast. Finally, flexibility of lipidomic features is proposed as a new parameter for the description of the physiological state of an organism

Mitochondria of the Yeasts Saccharomyces cerevisiae and Kluyveromyces lactis Contain Nuclear rDNA-Encoded Proteins

In eukaryotes, the nuclear ribosomal DNA (rDNA) is the source of the structural 18S, 5.8S and 25S rRNAs. In hemiascomycetous yeasts, the 25S rDNA sequence was described to lodge an antisense open reading frame (ORF) named TAR1 for Transcript Antisense to Ribosomal RNA. Here, we present the first immuno-detection and sub-cellular localization of the authentic product of this atypical yeast gene. Using specific antibodies against the predicted amino-acid sequence of the Saccharomyces cerevisiae TAR1 product, we detected the endogenous Tar1p polypeptides in S. cerevisiae (Sc) and Kluyveromyces lactis (Kl) species and found that both proteins localize to mitochondria. Protease and carbonate treatments of purified mitochondria further revealed that endogenous Sc Tar1p protein sub-localizes in the inner membrane in a Nin-Cout topology. Plasmid-versions of 5′ end or 3′ end truncated TAR1 ORF were used to demonstrate that neither the N-terminus nor the C-terminus of Sc Tar1p were required for its localization. Also, Tar1p is a presequence-less protein. Endogenous Sc Tar1p was found to be a low abundant protein, which is expressed in fermentable and non-fermentable growth conditions. Endogenous Sc TAR1 transcripts were also found low abundant and consistently 5′ flanking regions of TAR1 ORF exhibit modest promoter activity when assayed in a luciferase-reporter system. Using rapid amplification of cDNA ends (RACE) PCR, we also determined that endogenous Sc TAR1 transcripts possess heterogeneous 5′ and 3′ ends probably reflecting the complex expression of a gene embedded in actively transcribed rDNA sequence. Altogether, our results definitively ascertain that the antisense yeast gene TAR1 constitutes a functional transcription unit within the nuclear rDNA repeats

Growth landscape formed by perception and import of glucose in yeast

An important challenge in systems biology is to quantitatively describe microbial growth using a few measurable parameters that capture the essence of this complex phenomenon. Two key events at the cell membrane—extracellular glucose sensing and uptake—initiate the budding yeast’s growth on glucose. However, conventional growth models focus almost exclusively on glucose uptake. Here we present results from growth-rate experiments that cannot be explained by focusing on glucose uptake alone. By imposing a glucose uptake rate independent of the sensed extracellular glucose level, we show that despite increasing both the sensed glucose concentration and uptake rate, the cell’s growth rate can decrease or even approach zero. We resolve this puzzle by showing that the interaction between glucose perception and import, not their individual actions, determines the central features of growth, and characterize this interaction using a quantitative model. Disrupting this interaction by knocking out two key glucose sensors significantly changes the cell’s growth rate, yet uptake rates are unchanged. This is due to a decrease in burden that glucose perception places on the cells. Our work shows that glucose perception and import are separate and pivotal modules of yeast growth, the interaction of which can be precisely tuned and measured.National Institutes of Health (U.S.). Pioneer AwardNatural Sciences and Engineering Research Council of Canada (NSERC). Graduate Fellowshi

The Outcome of Phagocytic Cell Division with Infectious Cargo Depends on Single Phagosome Formation

Given that macrophages can proliferate and that certain microbes survive inside phagocytic cells, the question arises as to the post-mitotic distribution of microbial cargo. Using macrophage-like cells we evaluated the post-mitotic distribution of intracellular Cryptococcus yeasts and polystyrene beads by comparing experimental data to a stochastic model. For beads, the post-mitotic distribution was that expected from chance alone. However, for yeast cells the post-mitotic distribution was unequal, implying preferential sorting to one daughter cell. This mechanism for unequal distribution was phagosomal fusion, which effectively reduced the intracellular particle number. Hence, post-mitotic intracellular particle distribution is stochastic, unless microbial and/or host factors promote unequal distribution into daughter cells. In our system unequal cargo distribution appeared to benefit the microbe by promoting host cell exocytosis. Post-mitotic infectious cargo distribution is a new parameter to consider in the study of intracellular pathogens since it could potentially define the outcome of phagocytic-microbial interactions

Trf4 targets ncRNAs from telomeric and rDNA spacer regions and functions in rDNA copy number control

Trf4 is the poly(A) polymerase component of TRAMP4, which stimulates nuclear RNA degradation by the exosome. We report that in Saccharomyces cerevisiae strains lacking Trf4, cryptic transcripts are detected from regions of repressed chromatin at telomeres and the rDNA intergenic spacer region (IGS1-R), and at CEN3. Degradation of the IGS1-R transcript was reduced in strains lacking TRAMP components, the core exosome protein Mtr3 or the nuclear-specific exosome component Rrp6. IGS1-R has potential binding sites for the RNA-binding proteins Nrd1/Nab3, and was stabilized by mutation of Nrd1. IGS1-R passes through the replication fork barrier, a region required for rDNA copy number control. Strains lacking Trf4 showed sporadic changes in rDNA copy number, whereas loss of both Trf4 and either the histone deacetylase Sir2 or the topoisomerase Top1 caused dramatic loss of rDNA repeats. Chromatin immunoprecipitation analyses showed that Trf4 is co-transcriptionally recruited to IGS1-R, consistent with a direct role in rDNA stability. Co-transcriptional RNA binding by Trf4 may link RNA and DNA metabolism and direct immediate IGS1-R degradation by the exosome following transcription termination

Signaling Role of Fructose Mediated by FINS1/FBP in Arabidopsis thaliana

Sugars are evolutionarily conserved signaling molecules that regulate the growth and development of both unicellular and multicellular organisms. As sugar-producing photosynthetic organisms, plants utilize glucose as one of their major signaling molecules. However, the details of other sugar signaling molecules and their regulatory factors have remained elusive, due to the complexity of the metabolite and hormone interactions that control physiological and developmental programs in plants. We combined information from a gain-of-function cell-based screen and a loss-of-function reverse-genetic analysis to demonstrate that fructose acts as a signaling molecule in Arabidopsis thaliana. Fructose signaling induced seedling developmental arrest and interacted with plant stress hormone signaling in a manner similar to that of glucose. For fructose signaling responses, the plant glucose sensor HEXOKINASE1 (HXK1) was dispensable, while FRUCTOSE INSENSITIVE1 (FINS1), a putative FRUCTOSE-1,6-BISPHOSPHATASE, played a crucial role. Interestingly, FINS1 function in fructose signaling appeared to be independent of its catalytic activity in sugar metabolism. Genetic analysis further indicated that FINS1–dependent fructose signaling may act downstream of the abscisic acid pathway, in spite of the fact that HXK1–dependent glucose signaling works upstream of hormone synthesis. Our findings revealed that multiple layers of controls by fructose, glucose, and abscisic acid finely tune the plant autotrophic transition and modulate early seedling establishment after seed germination

Tinkering Evolution of Post-Transcriptional RNA Regulons: Puf3p in Fungi as an Example

Genome-wide studies of post-transcriptional mRNA regulation in model organisms indicate a “post-transcriptional RNA regulon” model, in which a set of functionally related genes is regulated by mRNA–binding RNAs or proteins. One well-studied post-transcriptional regulon by Puf3p functions in mitochondrial biogenesis in budding yeast. The evolution of the Puf3p regulon remains unclear because previous studies have shown functional divergence of Puf3p regulon targets among yeast, fruit fly, and humans. By analyzing evolutionary patterns of Puf3p and its targeted genes in forty-two sequenced fungi, we demonstrated that, although the Puf3p regulon is conserved among all of the studied fungi, the dedicated regulation of mitochondrial biogenesis by Puf3p emerged only in the Saccharomycotina clade. Moreover, the evolution of the Puf3p regulon was coupled with evolution of codon usage bias in down-regulating expression of genes that function in mitochondria in yeast species after genome duplication. Our results provide a scenario for how evolution like a tinker exploits pre-existing materials of a conserved post-transcriptional regulon to regulate gene expression for novel functional roles

The Function of MoGlk1 in Integration of Glucose and Ammonium Utilization in Magnaporthe oryzae

Hexokinases are conserved proteins functioning in glucose sensing and signaling. The rice blast fungus Magnaporthe oryzae contains several hexokinases, including MoHxk1 (hexokinase) and MoGlk1 (glucokinase) encoded respectively by MoHXK1 and MoGLK1 genes. The heterologous expression of MoGlk1 and MoHxk1 in Saccharomyces cerevisiae confirmed their conserved functions. Disruption of MoHXK1 resulted in growth reduction in medium containing fructose as the sole carbon source, whereas disruption of MoGLK1 did not cause the similar defect. However, the ΔMoglk1 mutant displayed decreased proton extrusion and a lower biomass in the presence of ammonium, suggesting a decline in the utilization of ammonium. Additionally, the MoGLK1 allele lacking catalytic activity restored growth to the ΔMoglk1 mutant. Moreover, the expression of MoPMA1 encoding a plasma membrane H+-ATPase decreased in the ΔMoglk1 mutant that can be suppressed by glucose and G-6-P. Thus, MoGlk1, but not MoHxk1, regulates ammonium utilization through a mechanism that is independent from its catalytic activity