scholarly journals Phospholipid flippases and Sfk1 are essential for the retention of ergosterol in the plasma membrane

2021 ◽  
pp. mbc.E20-11-0699
Author(s):  
Takuma Kishimoto ◽  
Tetsuo Mioka ◽  
Eriko Itoh ◽  
David E. Williams ◽  
Raymond J. Andersen ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Lipid Droplets ◽  
Eukaryotic Cells ◽  
Growth Defect ◽  
High Permeability ◽  
High Concentration ◽  
Phospholipid Asymmetry ◽  
Severe Growth Defect ◽  
Asymmetrically Distributed ◽  
Severe Growth

Sterols are important lipid components of the plasma membrane (PM) in eukaryotic cells, but it is unknown how the PM retains sterols at a high concentration. Phospholipids are asymmetrically distributed in the PM, and phospholipid flippases play an important role in generating this phospholipid asymmetry. Here, we provide evidence that phospholipid flippases are essential for retaining ergosterol in the PM of yeast. A mutant in three flippases, Dnf1-Lem3, Dnf2-Lem3, and Dnf3-Crf1, and a membrane protein, Sfk1, showed a severe growth defect. We recently identified Sfk1 as a PM protein involved in phospholipid asymmetry. The PM of this mutant showed high permeability and low density. Staining with the sterol probe filipin and the expression of a sterol biosensor revealed that ergosterol was not retained in the PM. Instead, ergosterol accumulated in an esterified form in lipid droplets. We propose that ergosterol is retained in the PM by the asymmetrical distribution of phospholipids and the action of Sfk1. Once phospholipid asymmetry is severely disrupted, sterols might be exposed on the cytoplasmic leaflet of the PM and actively transported to the endoplasmic reticulum by sterol transfer proteins.

scholarly journals Phospholipid flippases and Sfk1 are essential for the retention of ergosterol in the plasma membrane

2020 ◽  
Author(s):  
Takuma Kishimoto ◽  
Tetsuo Mioka ◽  
Eriko Itoh ◽  
David E. Williams ◽  
Raymond J. Andersen ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Lipid Droplets ◽  
Eukaryotic Cells ◽  
Growth Defect ◽  
High Permeability ◽  
High Concentration ◽  
Phospholipid Asymmetry ◽  
Severe Growth Defect ◽  
Asymmetrically Distributed ◽  
Severe Growth

AbstractSterols are important lipid components of the plasma membrane (PM) in eukaryotic cells, but it is unknown how the PM retains sterols at a high concentration. Phospholipids are asymmetrically distributed in the PM, and phospholipid flippases play an important role in generating this phospholipid asymmetry. Here, we provide evidence that phospholipid flippases are essential for retaining ergosterol in the PM of yeast. A mutant in three flippases, Dnf1-Lem3, Dnf2-Lem3, and Dnf3-Crf1, and a membrane protein, Sfk1, showed a severe growth defect. We recently identified Sfk1 as a PM protein involved in phospholipid asymmetry. The PM of this mutant showed high permeability and low density, and many nutrient transporters failed to localize to the PM. Staining with the sterol probe filipin and the expression of a sterol biosensor revealed that ergosterol was not retained in the PM. Instead, ergosterol accumulated in an esterified form in lipid droplets. We propose that ergosterol is retained in the PM by the asymmetrical distribution of phospholipids and the action of Sfk1. Once phospholipid asymmetry is severely disrupted, sterols might be exposed on the cytoplasmic leaflet of the PM and actively transported to the endoplasmic reticulum by sterol transfer proteins.

scholarly journals Trm11p and Trm112p Are both Required for the Formation of 2-Methylguanosine at Position 10 in Yeast tRNA

2005 ◽  
Vol 25 (11) ◽  
pp. 4359-4370 ◽  
Author(s):  
Suresh K. Purushothaman ◽  
Janusz M. Bujnicki ◽  
Henri Grosjean ◽  
Bruno Lapeyre
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Enzymatic Activity ◽  
Zinc Binding ◽  
Growth Defect ◽  
Putative Protein ◽  
Yeast Trna ◽  
Protein Methyltransferase ◽  
Severe Growth Defect ◽  
Severe Growth

ABSTRACT N 2 -Monomethylguanosine-10 (m2G10) and N 2 ,N 2 -dimethylguanosine-26 (m2 2G26) are the only two guanosine modifications that have been detected in tRNA from nearly all archaea and eukaryotes but not in bacteria. In Saccharomyces cerevisiae, formation of m2 2G26 is catalyzed by Trm1p, and we report here the identification of the enzymatic activity that catalyzes the formation of m2G10 in yeast tRNA. It is composed of at least two subunits that are associated in vivo: Trm11p (Yol124c), which is the catalytic subunit, and Trm112p (Ynr046w), a putative zinc-binding protein. While deletion of TRM11 has no detectable phenotype under laboratory conditions, deletion of TRM112 leads to a severe growth defect, suggesting that it has additional functions in the cell. Indeed, Trm112p is associated with at least four proteins: two tRNA methyltransferases (Trm9p and Trm11p), one putative protein methyltransferase (Mtc6p/Ydr140w), and one protein with a Rossmann fold dehydrogenase domain (Lys9p/Ynr050c). In addition, TRM11 interacts genetically with TRM1, thus suggesting that the absence of m2G10 and m2 2G26 affects tRNA metabolism or functioning.

scholarly journals bioA mutant of Mycobacterium tuberculosis shows severe growth defect and imparts protection against tuberculosis in guinea pigs

PLoS ONE ◽  
2017 ◽  
Vol 12 (6) ◽  
pp. e0179513 ◽  
Author(s):  
Ritika Kar ◽  
Prachi Nangpal ◽  
Shubhita Mathur ◽  
Swati Singh ◽  
Anil K. Tyagi
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Guinea Pigs ◽  
Growth Defect ◽  
Severe Growth Defect ◽  
Severe Growth

scholarly journals Dynamical localization of a thylakoid membrane binding protein is required for acquisition of photosynthetic competency

2017 ◽  
Author(s):  
Andrian Gutu ◽  
Frederick Chang ◽  
Erin K. O‘Shea
Keyword(s):  
Spatial Distribution ◽  
Thylakoid Membrane ◽  
Growth Conditions ◽  
Dynamical Localization ◽  
Growth Defect ◽  
Cell Periphery ◽  
Diffuse Fraction ◽  
Photosynthetic Complexes ◽  
Severe Growth Defect ◽  
Severe Growth

SUMMARYVipp1 is highly conserved and essential for photosynthesis, but its function is unclear as it does not participate directly in light-dependent reactions. We analyzed Vipp1 localization in live cyanobacterial cells and show that Vipp1 is highly dynamic, continuously exchanging between a diffuse fraction that is uniformly distributed throughout the cell and a punctate fraction that is concentrated at high curvature regions of the thylakoid located at the cell periphery. Experimentally perturbing the spatial distribution of Vipp1 by relocalizing it to the nucleoid causes a severe growth defect during the transition from non-photosynthetic (dark) to photosynthetic (light) growth. However, the same perturbation of Vipp1 in dark alone or light alone growth conditions causes no growth or thylakoid morphology defects. We propose that the punctuated dynamics of Vipp1 at the cell periphery in regions of high thylakoid curvature enable acquisition of photosynthetic competency, perhaps by facilitating biogenesis of photosynthetic complexes involved in light-dependent reactions of photosynthesis.

scholarly journals Insufficient levels of thenrdAB-encoded ribonucleotide reductase underlie the severe growth defect of the Δhda E. colistrain

2017 ◽  
Vol 104 (3) ◽  
pp. 377-399 ◽  
Author(s):  
Vignesh M. P. Babu ◽  
Mark Itsko ◽  
Jamie C. Baxter ◽  
Roel M. Schaaper ◽  
Mark D. Sutton
Keyword(s):  
Ribonucleotide Reductase ◽  
Growth Defect ◽  
Severe Growth Defect ◽  
Severe Growth

scholarly journals RelA/p65 Regulation of IκBβ

2005 ◽  
Vol 25 (12) ◽  
pp. 4956-4968 ◽  
Author(s):  
Erin Hertlein ◽  
Jingxin Wang ◽  
Katherine J. Ladner ◽  
Nadine Bakkar ◽  
Denis C. Guttridge
Keyword(s):  
Postnatal Development ◽  
26S Proteasome ◽  
Carboxyl Terminus ◽  
Growth Defect ◽  
Tight Control ◽  
Resistant Form ◽  
Specific Manner ◽  
Severe Growth Defect ◽  
Severe Growth ◽  
Hyperphosphorylated Form

ABSTRACT IκB inhibitor proteins are the primary regulators of NF-κB. In contrast to the defined regulatory interplay between NF-κB and IκBα, much less is known regarding the regulation of IκBβ by NF-κB. Here, we describe in detail the regulation of IκBβ by RelA/p65. Using p65 −/− fibroblasts, we show that IκBβ is profoundly reduced in these cells, but not in other NF-κB subunit knockouts. This regulation prevails during embryonic and postnatal development in a tissue-specific manner. Significantly, in both p65 −/− cells and tissues, IκBα is also reduced, but not nearly to the same extent as IκBβ, thus highlighting the degree to which IκBβ is dependent on p65. This dependence is based on the ability of p65 to stabilize IκBβ protein from the 26S proteasome, a process mediated in large part through the p65 carboxyl terminus. Furthermore, IκBβ was found to exist in both a basally phosphorylated and a hyperphosphorylated form. While the hyperphosphorylated form is less abundant, it is also more stable and less dependent on p65 and its carboxyl domain. Finally, we show that in p65 −/− fibroblasts, expression of a proteolysis-resistant form of IκBβ, but not IκBα, causes a severe growth defect associated with apoptosis. Based on these findings, we propose that tight control of IκBβ protein by p65 is necessary for the maintenance of cellular homeostasis.

scholarly journals Human mutation D157G in ferroportin leads to hepcidin-independent binding of Jak2 and ferroportin down-regulation

Blood ◽  
2010 ◽  
Vol 115 (14) ◽  
pp. 2956-2959 ◽  
Author(s):  
Ivana De Domenico ◽  
Eric Lo ◽  
Diane M. Ward ◽  
Jerry Kaplan
Keyword(s):  
Iron Homeostasis ◽  
Fluorescent Protein ◽  
Transferrin Saturation ◽  
High Serum ◽  
Growth Defect ◽  
Down Regulation ◽  
Severe Growth Defect ◽  
Human Mutation ◽  
Green Fluorescent ◽  
Severe Growth

Abstract Mutations in the iron exporter ferroportin (Fpn) result in iron overload in macrophages or hepatocytes depending upon the mutation. Patients with Fpn mutation D157G show high serum ferritin and normal to slightly elevated transferrin saturation. Here, we show that Fpn(D157G)–green fluorescent protein (GFP) is down-regulated independent of hepcidin, and that this down-regulation is due to the constitutive binding of Jak2 and Fpn phosphorylation. Expression of Fpn(D157G)-GFP in Danio rerio results in a severe growth defect, which can be rescued by iron supplementation. These results identify a hepcidin-independent regulation of Fpn that can result in alterations in iron homeostasis.

scholarly journals The nucleolar DExD/H protein Hel66 is involved in ribosome biogenesis in Trypanosoma brucei

2021 ◽  
Author(s):  
Majeed Bakari-Soale ◽  
Nonso Josephat Ikenge ◽  
Marion Scheibe ◽  
Falk Butter ◽  
Nicola Gail Jones ◽  
...  
Keyword(s):  
Ribosomal Rna ◽  
Ribosomal Proteins ◽  
Ribosome Biogenesis ◽  
Growth Defect ◽  
Rrna Processing ◽  
Ribosomal Subunits ◽  
Comprehensive Picture ◽  
Severe Growth Defect ◽  
H Protein ◽  
Severe Growth

The biosynthesis of ribosomes is a complex cellular process involving ribosomal RNA, ribosomal proteins and several further trans-acting factors. DExD/H box proteins constitute the largest family of trans-acting protein factors involved in this process. Several members of this protein family have been directly implicated in ribosome biogenesis in yeast. In trypanosomes, ribosome biogenesis differs in several features from the process described in yeast. Here, we have identified the DExD/H box helicase Hel66 as being involved in ribosome biogenesis. The protein is unique to Kinetoplastida, localises to the nucleolus and its depletion via RNAi caused a severe growth defect. Loss of the protein resulted in a decrease of global translation and accumulation of rRNA processing intermediates for both the small and large ribosomal subunits. Only a few factors involved in trypanosome rRNA biogenesis have been described so far and our findings contribute to gaining a more comprehensive picture of this essential process.

scholarly journals Severe growth defect in a Schizosaccharomyces pombe mutant defective in intron lariat degradation.

1997 ◽  
Vol 17 (2) ◽  
pp. 809-818 ◽  
Author(s):  
K Nam ◽  
G Lee ◽  
J Trambley ◽  
S E Devine ◽  
J D Boeke
Keyword(s):  
Cell Growth ◽  
Schizosaccharomyces Pombe ◽  
Rna Degradation ◽  
Growth Defect ◽  
Null Mutant ◽  
Rate Limiting Step ◽  
Genes Encoding ◽  
Severe Growth Defect ◽  
Rate Limiting ◽  
Severe Growth

The cDNAs and genes encoding the intron lariat-debranching enzyme were isolated from the nematode Caenorhabditis elegans and the fission yeast Schizosaccharomyces pombe based on their homology with the Saccharomyces cerevisiae gene. The cDNAs were shown to be functional in an interspecific complementation experiment; they can complement an S. cerevisiae dbr1 null mutant. About 2.5% of budding yeast S. cerevisiae genes have introns, and the accumulation of excised introns in a dbr1 null mutant has little effect on cell growth. In contrast, many S. pombe genes contain introns, and often multiple introns per gene, so that S. pombe is estimated to contain approximately 40 times as many introns as S. cerevisiae. The S. pombe dbr1 gene was disrupted and shown to be nonessential. Like the S. cerevisiae mutant, the S. pombe null mutant accumulated introns to high levels, indicating that intron lariat debranching represents a rate-limiting step in intron degradation in both species. Unlike the S. cerevisiae mutant, the S. pombe dbr1::leu1+ mutant had a severe growth defect and exhibited an aberrant elongated cell shape in addition to an intron accumulation phenotype. The growth defect of the S. pombe dbr1::leu1+ strain suggests that debranching activity is critical for efficient intron RNA degradation and that blocking this pathway interferes with cell growth.

scholarly journals The Nucleolar DExD/H Protein Hel66 is Involved in Ribosome Biogenesis in Trypanosoma Brucei

2021 ◽  
Author(s):  
Majeed Bakari-Soale ◽  
Nonso Josephat Ikenga ◽  
Marion Scheibe ◽  
Falk Butter ◽  
Nicola Gail Jones ◽  
...  
Keyword(s):  
Ribosomal Rna ◽  
Ribosomal Proteins ◽  
Ribosome Biogenesis ◽  
Growth Defect ◽  
Rrna Processing ◽  
Ribosomal Subunits ◽  
Comprehensive Picture ◽  
Severe Growth Defect ◽  
H Protein ◽  
Severe Growth

Abstract The biosynthesis of ribosomes is a complex cellular process involving ribosomal RNA, ribosomal proteins and several further trans-acting factors. DExD/H box proteins constitute the largest family of trans-acting protein factors involved in this process. Several members of this protein family have been directly implicated in ribosome biogenesis in yeast. In trypanosomes, ribosome biogenesis differs in several features from the process described in yeast. Here, we have identified the DExD/H box helicase Hel66 as being involved in ribosome biogenesis. The protein is unique to Kinetoplastida, localises to the nucleolus and its depletion via RNAi caused a severe growth defect. Loss of the protein resulted in a decrease of global translation and accumulation of rRNA processing intermediates for both the small and large ribosomal subunits. Only a few factors involved in trypanosome rRNA biogenesis have been described so far and our findings contribute to gaining a more comprehensive picture of this essential process.

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