scholarly journals Identification of Pex34p as a component of the peroxisomal de novo biogenesis machinery in yeast

2021 ◽  
Author(s):  
Juliane Radke ◽  
Shirisha Nagotu ◽  
Wolfgang Girzalsky ◽  
Anirban Chakraborty ◽  
Markus Deckers ◽  
...  
Keyword(s):  
Fatty Acids ◽  
Environmental Changes ◽  
De Novo ◽  
Yeast Cells ◽  
Dependent Manner ◽  
Peroxisomal Membrane ◽  
Expression Screening ◽  
A Cell ◽  
Conditional Inhibition ◽  
Yeast Mutants

Cells can regulate the abundance and composition of peroxisomes to adapt to environmental changes. In the bakers yeast, S. cerevisiae, peroxisomes represent the only site for degradation of fatty acids. Hence, it is not surprising that growth of yeast cells on oleic acid results in a massive proliferation of peroxisomes. New peroxisomes can form either by division of pre-existing peroxisomes or de novo in a Pex25p-dependent process with the involvement of the Endoplasmic Reticulum (ER). In search for further factors involved in de novo formation of peroxisomes, we screened nearly 6,000 yeast mutants that were depleted of peroxisomes by conditional inhibition of PEX19 expression. Screening the mutants for the reappearance of peroxisomes upon expression of PEX19 identified Pex34p, in addition to the well-known component Pex25p, as crucial determinants for de novo biogenesis. Pex34p interacts with Pex19p and with different Peroxisomal Membrane Proteins (PMPs) in a PEX19-dependent manner. Depletion of Pex34p results in reduced numbers of import-competent peroxisomes formed de novo and Pex3p is partly retained and distributed in ER-like structures. We suggest that Pex25p and Pex34p are both required to maintain peroxisome number in a cell and that they perform non-redundant roles in the de novo formation of peroxisomes.

scholarly journals Peroxisome Maintenance Depends on De Novo Peroxisome Formation in Yeast Mutants Defective in Peroxisome Fission and Inheritance

2019 ◽  
Vol 20 (16) ◽  
pp. 4023 ◽  
Author(s):  
Justyna P. Wróblewska ◽  
Ida J. van der Klei
Keyword(s):  
De Novo ◽  
Hansenula Polymorpha ◽  
Yeast Cells ◽  
Wild Type ◽  
Double Deletion ◽  
Mother Cells ◽  
Yeast Mutants ◽  
Rescue Mechanism ◽  
Wild Type Yeast ◽  
In Cells

There is an ongoing debate on how peroxisomes form: by growth and fission of pre-existing peroxisomes or de novo from another membrane. It has been proposed that, in wild type yeast cells, peroxisome fission and careful segregation of the organelles over mother cells and buds is essential for organelle maintenance. Using live cell imaging we observed that cells of the yeast Hansenula polymorpha, lacking the peroxisome fission protein Pex11, still show peroxisome fission and inheritance. Also, in cells of mutants without the peroxisome inheritance protein Inp2 peroxisome segregation can still occur. In contrast, peroxisome fission and inheritance were not observed in cells of a pex11 inp2 double deletion strain. In buds of cells of this double mutant, new organelles likely appear de novo. Growth of pex11 inp2 cells on methanol, a growth substrate that requires functional peroxisomes, is retarded relative to the wild type control. Based on these observations we conclude that in H. polymorpha de novo peroxisome formation is a rescue mechanism, which is less efficient than organelle fission and inheritance to maintain functional peroxisomes.

scholarly journals Bacterial Toxins Induce Sustained mRNA Expression of the Silencing Transcription Factor klf2 via Inactivation of RhoA and Rhophilin 1

2009 ◽  
Vol 77 (12) ◽  
pp. 5583-5592 ◽  
Author(s):  
Kristina Dach ◽  
Josip Zovko ◽  
Michael Hogardt ◽  
Isabel Koch ◽  
Katrin van Erp ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Pseudomonas Aeruginosa ◽  
Transcription Factor ◽  
Serum Response Factor ◽  
De Novo ◽  
Virulence Plasmid ◽  
Dependent Manner ◽  
Expression Screening ◽  
Mrna Induction ◽  
Serum Response

ABSTRACTYersiniae bearing theYersiniavirulence plasmid pYV impact the transcriptome of J774A.1 macrophage-like cells in two distinct ways: (i) by suppressing, in aYersiniaouter protein P (YopP)-dependent manner, the induction of inflammatory response genes and (ii) by mRNA induction of the silencing transcription factorklf2. Here we show thatklf2induction byYersinia enterocoliticaoccurs in several cell lines of macrophage and squamous and upper gastrointestinal epithelial origin as well as in bone marrow-derived dendritic cells. Several strains ofPseudomonas aeruginosaandStaphylococcus aureusare equally effective asY. enterocoliticain inducingklf2expression. Screening of mutant strains or incubation with recombinant toxins identified the rho-inactivating toxins YopT fromYersiniaspp., ExoS fromPseudomonas aeruginosa, EDIN-B fromStaphylococcus aureus, and C3bot fromClostridium botulinumas bacterial inducers ofklf2mRNA.klf2mRNA induction by these toxins does not require de novo protein synthesis. Serum response factor or actin depolymerization does not seem to be involved in regulatingklf2expression in response to bacterial infection. Instead, short hairpin RNA-mediated inactivation of RhoA and its effector rhophilin 1 is sufficient to induce long-termklf2expression. Thus, bacteria exploit the RhoA-rhophilin signaling cascade to mediate sustained expression of the immunosuppressive transcription factorklf2.

scholarly journals Small-Molecule Modulators of Listeria monocytogenes Biofilm Development

2011 ◽  
Vol 78 (5) ◽  
pp. 1454-1465 ◽  
Author(s):  
Uyen T. Nguyen ◽  
Iwona B. Wenderska ◽  
Matthew A. Chong ◽  
Kalinka Koteva ◽  
Gerard D. Wright ◽  
...  
Keyword(s):  
Fatty Acids ◽  
Listeria Monocytogenes ◽  
Protein Kinase ◽  
Biofilm Formation ◽  
De Novo ◽  
Saturated Fatty Acids ◽  
Antibiofilm Activity ◽  
Dependent Manner ◽  
Content Type ◽  
Biofilm Development

ABSTRACTListeria monocytogenesis an important food-borne pathogen whose ability to form disinfectant-tolerant biofilms on a variety of surfaces presents a food safety challenge for manufacturers of ready-to-eat products. We developed here a high-throughput biofilm assay forL. monocytogenesand, as a proof of principle, used it to screen an 80-compound protein kinase inhibitor library to identify molecules that perturb biofilm development. The screen yielded molecules toxic to multiple strains ofListeriaat micromolar concentrations, as well as molecules that decreased (≤50% of vehicle control) or increased (≥200%) biofilm formation in a dose-dependent manner without affecting planktonic cell density. Toxic molecules—including the protein kinase C antagonist sphingosine—had antibiofilm activity at sub-MIC concentrations. Structure-activity studies of the biofilm inhibitory compound palmitoyl-d,l-carnitine showed that whileListeriabiofilm formation was inhibited with a 50% inhibitory concentration of 5.85 ± 0.24 μM,d,l-carnitine had no effect, whereas palmitic acid had stimulatory effects. Saturated fatty acids between C9:0and C14:0wereListeriabiofilm inhibitors, whereas fatty acids of C16:0or longer were stimulators, showing chain length specificity.De novo-synthesized short-chain acyl carnitines were less effective biofilm inhibitors than the palmitoyl forms. These molecules, whose activities against bacteria have not been previously established, are both useful probes ofL. monocytogenesbiology and promising leads for the further development of antibiofilm strategies.

scholarly journals Lipids and Trehalose Actively Cooperate in Heat Stress Management of Schizosaccharomyces pombe

2021 ◽  
Vol 22 (24) ◽  
pp. 13272
Author(s):  
Mária Péter ◽  
Péter Gudmann ◽  
Zoltán Kóta ◽  
Zsolt Török ◽  
László Vígh ◽  
...  
Keyword(s):  
Fatty Acids ◽  
Heat Stress ◽  
Schizosaccharomyces Pombe ◽  
De Novo ◽  
Acyl Chain ◽  
Membrane Lipid ◽  
Yeast Cells ◽  
Storage Lipids ◽  
Acyl Chain Length

Homeostatic maintenance of the physicochemical properties of cellular membranes is essential for life. In yeast, trehalose accumulation and lipid remodeling enable rapid adaptation to perturbations, but their crosstalk was not investigated. Here we report about the first in-depth, mass spectrometry-based lipidomic analysis on heat-stressed Schizosaccharomyces pombe mutants which are unable to synthesize (tps1Δ) or degrade (ntp1Δ) trehalose. Our experiments provide data about the role of trehalose as a membrane protectant in heat stress. We show that under conditions of trehalose deficiency, heat stress induced a comprehensive, distinctively high-degree lipidome reshaping in which structural, signaling and storage lipids acted in concert. In the absence of trehalose, membrane lipid remodeling was more pronounced and increased with increasing stress dose. It could be characterized by decreasing unsaturation and increasing acyl chain length, and required de novo synthesis of stearic acid (18:0) and very long-chain fatty acids to serve membrane rigidification. In addition, we detected enhanced and sustained signaling lipid generation to ensure transient cell cycle arrest as well as more intense triglyceride synthesis to accommodate membrane lipid-derived oleic acid (18:1) and newly synthesized but unused fatty acids. We also demonstrate that these changes were able to partially substitute for the missing role of trehalose and conferred measurable stress tolerance to fission yeast cells.

scholarly journals Degradation of human thymidine kinase is dependent on serine-13 phosphorylation: involvement of the SCF-mediated pathway

2003 ◽  
Vol 370 (1) ◽  
pp. 265-273 ◽  
Author(s):  
Po-Yuan KE ◽  
Che-Chuan YANG ◽  
I-Chun TSAI ◽  
Zee-Fen CHANG
Keyword(s):  
Cell Cycle ◽  
Protein Degradation ◽  
Thymidine Kinase ◽  
Yeast Cells ◽  
Temperature Sensitive ◽  
Dependent Manner ◽  
Proteolytic Pathway ◽  
A Cell ◽  
The Stability ◽  
Cycle Dependent

The expression level of human thymidine kinase (hTK) is regulated in a cell-cycle-dependent manner. One of the mechanisms responsible for the fluctuation of TK expression in the cell cycle can be attributed to protein degradation during mitosis. Given the facts that cell-cycle-dependent proteolysis is highly conserved in all eukaryotes and yeast cells are an excellent model system for protein-degradation study, here we report on the use of Saccharomyces cerevisiae and Schizosaccharomyces pombe to investigate the degradation signal and mechanism required for hTK degradation. We found that the stability of hTK is significantly reduced in mitotic yeasts. Previously, we have observed that Ser-13 is the site of mitotic phosphorylation of hTK in HeLa cells [Chang, Huang and Chi (1998) J. Biol. Chem. 273, 12095—12100]. Here, we further provide evidence that the replacement of Ser-13 by Ala (S13A) renders hTK stable in S. pombe and S. cerevisiae. Most interestingly, we demonstrated that degradation of hTK is impaired in S. cerevisiae carrying a temperature-sensitive mutation in the proteasomal gene pre1-1 or the Skp1-Cullin-1/CDC53-F-box (SCF) complex gene cdc34 or cdc53, suggesting the contribution of the SCF-mediated pathway in hTK degradation. As phosphorylation is a prerequisite signal for SCF recognition, our results implied that phosphorylation of Ser-13 probably contributes to the degradation signal for hTK via the SCF-mediated proteolytic pathway.

Inhibition of the Plastidic Pyruvate Dehydrogenase Complex in Isolated Plastids of Oat

1994 ◽  
Vol 49 (7-8) ◽  
pp. 421-426 ◽  
Author(s):  
Andrea Golz ◽  
Hartmut K. Lichtenthaler
Keyword(s):  
Fatty Acids ◽  
Fatty Acid ◽  
Pyruvate Dehydrogenase ◽  
Fatty Acid Biosynthesis ◽  
De Novo ◽  
Pyruvate Dehydrogenase Complex ◽  
Dependent Manner ◽  
Acid Biosynthesis ◽  
Acetyl Coa ◽  
Dehydrogenase Complex

The activity of the plastidic pyruvate dehydrogenase complex (pPDHC) is one source of acetyl-CoA in plastids of higher plants needed for de novo fatty acid biosynthesis. This plastidic enzyme reaction is specifically inhibited by acetylmethylphosphinate (AMPI), a com ­ pound which had hitherto been known only as an inhibitor of the mitochondrial pyruvate dehydrogenase complex (mPDHC). In the test system of isolated intact oat plastids (Avena sativa) [2-14C]pyruvate was used for de novo fatty acid biosynthesis. The incorporation of label from [2-14C]pyruvate in fatty acids was inhibited by AMPI in a dose-dependent manner. The inhibition rose with increasing preincubation time of plastids with the inhibitor. I50 values for the inhibition of de novo fatty acid biosynthesis from [2-14C]pyruvate by AMPI for iso­lated etioplasts and chloroplasts were 4.5 and 80 μm , respectively. The activity of the pPDHC decreased during greening of oat seedlings, as is seen from the decreasing incorporation of [2-14C]pyruvate into fatty acids during the light-induced transformation of etioplasts into chloroplasts. In contrast to the decreasing pPDHC activity, the activity of the plastidic acetyl-C oA synthetase (ACS), which transfers acetate to acetyl-CoA, rose parallel to the transfor­mation of etioplasts into chloroplasts. During the assay time of 20 min we could not detect an incorporation of radiolabel from pyruvate or acetate into β-carotene or any other carotenoid

scholarly journals A Bni4-Glc7 Phosphatase Complex That Recruits Chitin Synthase to the Site of Bud Emergence

2003 ◽  
Vol 14 (1) ◽  
pp. 26-39 ◽  
Author(s):  
Lukasz Kozubowski ◽  
Heather Panek ◽  
Ashley Rosenthal ◽  
Andrew Bloecher ◽  
Douglas J. DeMarini ◽  
...  
Keyword(s):  
Chitin Synthase ◽  
Yeast Cells ◽  
Regulatory Subunit ◽  
Dependent Manner ◽  
Yeast Saccharomyces Cerevisiae ◽  
Bud Emergence ◽  
Bud Neck ◽  
A Cell ◽  
Cycle Dependent

Bni4 is a scaffold protein in the yeast Saccharomyces cerevisiae that tethers chitin synthase III to the bud neck by interacting with septin neck filaments and with Chs4, a regulatory subunit of chitin synthase III. We show herein that Bni4 is also a limiting determinant for the targeting of the type 1 serine/threonine phosphatase (Glc7) to the bud neck. Yeast cells containing a Bni4 variant that fails to associate with Glc7 fail to tether Chs4 to the neck, due in part to the failure of Bni4V831A/F833A to localize properly. Conversely, the Glc7-129 mutant protein fails to bind Bni4 properly and glc7-129 mutants exhibit reduced levels of Bni4 at the bud neck. Bni4 is phosphorylated in a cell cycle-dependent manner and Bni4V831A/F833A is both hyperphosphorylated and mislocalized in vivo. Yeast cells lacking the protein kinase Hsl1 exhibit increased levels of Bni4-GFP at the bud neck. GFP-Chs4 does not accumulate at the incipient bud site in either a bni4::TRP1 or abni4 V831A/F833A mutant but does mobilize to the neck at cytokinesis. Together, these results indicate that the formation of the Bni4-Glc7 complex is required for localization to the site of bud emergence and for subsequent targeting of chitin synthase.

scholarly journals Targeting of Tsc13p to Nucleus–Vacuole Junctions: A Role for Very-Long-Chain Fatty Acids in the Biogenesis of Microautophagic Vesicles

2005 ◽  
Vol 16 (9) ◽  
pp. 3987-3998 ◽  
Author(s):  
Erik Kvam ◽  
Kenneth Gable ◽  
Teresa M. Dunn ◽  
David S. Goldfarb
Keyword(s):  
Fatty Acids ◽  
Fatty Acid ◽  
De Novo ◽  
Acid Synthesis ◽  
Dependent Manner ◽  
Long Chain Fatty Acids ◽  
Vacuole Membrane ◽  
Mutant Cells ◽  
Long Chain ◽  
Chain Fatty Acids

TSC13 is required for the biosynthesis of very-long-chain fatty acids (VLCFAs) in yeast. Tsc13p is a polytopic endoplasmic reticulum (ER) membrane protein that accumulates at nucleus–vacuole (NV) junctions, which are formed through Velcro-like interactions between Nvj1p in the perinuclear ER and Vac8p on the vacuole membrane. NV junctions mediate piecemeal microautophagy of the nucleus (PMN), during which bleb-like portions of the nucleus are extruded into invaginations of the vacuole membrane and degraded in the vacuole lumen. We report that Tsc13p is sequestered into NV junctions from the peripheral ER through Vac8p-independent interactions with Nvj1p. During nutrient limitation, Tsc13p is incorporated into PMN vesicles in an Nvj1p-dependent manner. The lumenal diameters of PMN blebs and vesicles are significantly reduced in tsc13-1 and tsc13-1 elo3-Δ mutant cells. PMN structures are also smaller in cells treated with cerulenin, an inhibitor of de novo fatty acid synthesis and elongation. The targeting of Tsc13p-GFP into NV junctions is perturbed by cerulenin, suggesting that its binding to Nvj1p depends on the availability of fatty acid substrates. These results indicate that Nvj1p retains and compartmentalizes Tsc13p at NV junctions and that VLCFAs contribute to the normal biogenesis of trilaminar PMN structures in yeast.

scholarly journals Cell cycle–dependent spatial segregation of telomerase from sites of DNA damage

2017 ◽  
Vol 216 (8) ◽  
pp. 2355-2371 ◽  
Author(s):  
Faissal Ouenzar ◽  
Maxime Lalonde ◽  
Hadrien Laprade ◽  
Geneviève Morin ◽  
Franck Gallardo ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dna Repair ◽  
Single Molecule ◽  
De Novo ◽  
Dependent Manner ◽  
Dna Double Strand Breaks ◽  
Yeast Telomerase ◽  
A Cell ◽  
Spatial Exclusion ◽  
Cycle Dependent

Telomerase can generate a novel telomere at DNA double-strand breaks (DSBs), an event called de novo telomere addition. How this activity is suppressed remains unclear. Combining single-molecule imaging and deep sequencing, we show that the budding yeast telomerase RNA (TLC1 RNA) is spatially segregated to the nucleolus and excluded from sites of DNA repair in a cell cycle–dependent manner. Although TLC1 RNA accumulates in the nucleoplasm in G1/S, Pif1 activity promotes TLC1 RNA localization in the nucleolus in G2/M. In the presence of DSBs, TLC1 RNA remains nucleolar in most G2/M cells but accumulates in the nucleoplasm and colocalizes with DSBs in rad52Δ cells, leading to de novo telomere additions. Nucleoplasmic accumulation of TLC1 RNA depends on Cdc13 localization at DSBs and on the SUMO ligase Siz1, which is required for de novo telomere addition in rad52Δ cells. This study reveals novel roles for Pif1, Rad52, and Siz1-dependent sumoylation in the spatial exclusion of telomerase from sites of DNA repair.

Spindle scaling mechanisms

2020 ◽  
Vol 64 (2) ◽  
pp. 383-396
Author(s):  
Lara K. Krüger ◽  
Phong T. Tran
Keyword(s):  
Cell Size ◽  
Spindle Assembly ◽  
Dependent Manner ◽  
Post Translational Modification ◽  
Size Dependent ◽  
A Cell ◽  
Chromosome Separation ◽  
Spindle Elongation ◽  
Protein Gradients ◽  
Spindle Structure

Abstract The mitotic spindle robustly scales with cell size in a plethora of different organisms. During development and throughout evolution, the spindle adjusts to cell size in metazoans and yeast in order to ensure faithful chromosome separation. Spindle adjustment to cell size occurs by the scaling of spindle length, spindle shape and the velocity of spindle assembly and elongation. Different mechanisms, depending on spindle structure and organism, account for these scaling relationships. The limited availability of critical spindle components, protein gradients, sequestration of spindle components, or post-translational modification and differential expression levels have been implicated in the regulation of spindle length and the spindle assembly/elongation velocity in a cell size-dependent manner. In this review, we will discuss the phenomenon and mechanisms of spindle length, spindle shape and spindle elongation velocity scaling with cell size.

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