Guanosine diphosphatase is required for protein and sphingolipid glycosylation in the Golgi lumen of Saccharomyces cerevisiae

C Abeijon; K Yanagisawa; EC Mandon; A Häusler; K Moremen; CB Hirschberg; PW Robbins

doi:10.1083/jcb.122.2.307

Guanosine diphosphatase is required for protein and sphingolipid glycosylation in the Golgi lumen of Saccharomyces cerevisiae

The Journal of Cell Biology ◽

10.1083/jcb.122.2.307 ◽

1993 ◽

Vol 122 (2) ◽

pp. 307-323 ◽

Cited By ~ 123

Author(s):

C Abeijon ◽

K Yanagisawa ◽

EC Mandon ◽

A Häusler ◽

K Moremen ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Golgi Apparatus ◽

Critical Role ◽

Carboxypeptidase Y ◽

Null Mutation ◽

Nucleotide Sugar ◽

Additional Nucleotide ◽

Nucleoside Monophosphates ◽

Nucleoside Diphosphatase

Current models for nucleotide sugar use in the Golgi apparatus predict a critical role for the lumenal nucleoside diphosphatase. After transfer of sugars to endogenous macromolecular acceptors, the enzyme converts nucleoside diphosphates to nucleoside monophosphates which in turn exit the Golgi lumen in a coupled antiporter reaction, allowing entry of additional nucleotide sugar from the cytosol. To test this model, we cloned the gene for the S. cerevisiae guanosine diphosphatase and constructed a null mutation. This mutation should reduce the concentrations of GDP-mannose and GMP and increase the concentration of GDP in the Golgi lumen. The alterations should in turn decrease mannosylation of proteins and lipids in this compartment. In fact, we found a partial block in O- and N-glycosylation of proteins such as chitinase and carboxypeptidase Y and underglycosylation of invertase. In addition, mannosylinositolphosphorylceramide levels were drastically reduced.

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A Genome-Wide Screen in Saccharomyces cerevisiae Reveals a Critical Role for Oxidative Phosphorylation in Cellular Tolerance to Lithium Hexafluorophosphate

Cells ◽

10.3390/cells10040888 ◽

2021 ◽

Vol 10 (4) ◽

pp. 888

Author(s):

Xuejiao Jin ◽

Jie Zhang ◽

Tingting An ◽

Huihui Zhao ◽

Wenhao Fu ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Oxidative Phosphorylation ◽

Cellular Response ◽

Critical Role ◽

Lithium Ion ◽

Deletion Mutants ◽

High Concentration ◽

Genome Wide ◽

A Genome ◽

Lithium Hexafluorophosphate

Lithium hexafluorophosphate (LiPF6) is one of the leading electrolytes in lithium-ion batteries, and its usage has increased tremendously in the past few years. Little is known, however, about its potential environmental and biological impacts. In order to improve our understanding of the cytotoxicity of LiPF6 and the specific cellular response mechanisms to it, we performed a genome-wide screen using a yeast (Saccharomyces cerevisiae) deletion mutant collection and identified 75 gene deletion mutants that showed LiPF6 sensitivity. Among these, genes associated with mitochondria showed the most enrichment. We also found that LiPF6 is more toxic to yeast than lithium chloride (LiCl) or sodium hexafluorophosphate (NaPF6). Physiological analysis showed that a high concentration of LiPF6 caused mitochondrial damage, reactive oxygen species (ROS) accumulation, and ATP content changes. Compared with the results of previous genome-wide screening for LiCl-sensitive mutants, we found that oxidative phosphorylation-related mutants were specifically hypersensitive to LiPF6. In these deletion mutants, LiPF6 treatment resulted in higher ROS production and reduced ATP levels, suggesting that oxidative phosphorylation-related genes were important for counteracting LiPF6-induced toxicity. Taken together, our results identified genes specifically involved in LiPF6-modulated toxicity, and demonstrated that oxidative stress and ATP imbalance maybe the driving factors in governing LiPF6-induced toxicity.

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The Spindle Checkpoint of the Yeast Saccharomyces cerevisiae Requires Kinetochore Function and Maps to the CBF3 Domain

Genetics ◽

10.1093/genetics/157.4.1493 ◽

2001 ◽

Vol 157 (4) ◽

pp. 1493-1502

Author(s):

Richard D Gardner ◽

Atasi Poddar ◽

Chris Yellman ◽

Penny A Tavormina ◽

M Cristina Monteagudo ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Critical Role ◽

Spindle Checkpoint ◽

Essential Genes ◽

Gene Fusions ◽

Yeast Saccharomyces Cerevisiae ◽

Checkpoint Signaling ◽

One Step ◽

Diploid Cells ◽

Kinetochore Function

Abstract We have measured the activity of the spindle checkpoint in null mutants lacking kinetochore activity in the yeast Saccharomyces cerevisiae. We constructed deletion mutants for nonessential genes by one-step gene replacements. We constructed heterozygous deletions of one copy of essential genes in diploid cells and purified spores containing the deletion allele. In addition, we made gene fusions for three essential genes to target the encoded proteins for proteolysis (degron alleles). We determined that Ndc10p, Ctf13p, and Cep3p are required for checkpoint activity. In contrast, cells lacking Cbf1p, Ctf19p, Mcm21p, Slk19p, Cse4p, Mif2p, Mck1p, and Kar3p are checkpoint proficient. We conclude that the kinetochore plays a critical role in checkpoint signaling in S. cerevisiae. Spindle checkpoint activity maps to a discreet domain within the kinetochore and depends on the CBF3 protein complex.

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Comparisons of Mutants Lacking the Golgi UDP-Galactose or GDP-Mannose Transporters Establish that Phosphoglycans Are Important for Promastigote but Not Amastigote Virulence in Leishmania major

Infection and Immunity ◽

10.1128/iai.00735-07 ◽

2007 ◽

Vol 75 (9) ◽

pp. 4629-4637 ◽

Cited By ~ 36

Author(s):

Althea A. Capul ◽

Suzanne Hickerson ◽

Tamara Barron ◽

Salvatore J. Turco ◽

Stephen M. Beverley

Keyword(s):

Golgi Apparatus ◽

Protective Immunity ◽

Leishmania Major ◽

Protozoan Parasite ◽

Macrophage Infection ◽

Nucleotide Sugar ◽

Infectious Cycle ◽

Null Mutants

ABSTRACT Abundant surface Leishmania phosphoglycans (PGs) containing [Gal(β1,4)Man(α1-PO4)]-derived repeating units are important at several points in the infectious cycle of this protozoan parasite. PG synthesis requires transport of activated nucleotide-sugar precursors from the cytoplasm to the Golgi apparatus. Correspondingly, null mutants of the L. major GDP-mannose transporter LPG2 lack PGs and are severely compromised in macrophage survival and induction of acute pathology in susceptible mice, yet they are able to persist indefinitely and induce protective immunity. However, lpg2 − L. mexicana amastigotes similarly lacking PGs but otherwise normal in known glycoconjugates remain able to induce acute pathology. To explore this further, we tested the infectivity of a new PG-null L. major mutant, which is inactivated in the two UDP-galactose transporter genes LPG5A and LPG5B. Surprisingly this mutant did not recapitulate the phenotype of L. major lpg2 −, instead resembling the L. major lipophosphoglycan-deficient lpg1 − mutant. Metacyclic lpg5A −/lpg5B − promastigotes showed strong defects in the initial steps of macrophage infection and survival. However, after a modest delay, the lpg5A − /lpg5B − mutant induced lesion pathology in infected mice, which thereafter progressed normally. Amastigotes recovered from these lesions were fully infective in mice and in macrophages despite the continued absence of PGs. This suggests that another LPG2-dependent metabolite is responsible for the L. major amastigote virulence defect, although further studies ruled out cytoplasmic mannans. These data thus resolve the distinct phenotypes seen among lpg2 − Leishmania species by emphasizing the role of glycoconjugates other than PGs in amastigote virulence, while providing further support for the role of PGs in metacyclic promastigote virulence.

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Structure and molecular analysis of RGR1, a gene required for glucose repression of Saccharomyces cerevisiae.

Molecular and Cellular Biology ◽

10.1128/mcb.10.8.4130 ◽

1990 ◽

Vol 10 (8) ◽

pp. 4130-4138 ◽

Cited By ~ 58

Author(s):

A Sakai ◽

Y Shimizu ◽

S Kondou ◽

T Chibazakura ◽

F Hishinuma

Keyword(s):

Saccharomyces Cerevisiae ◽

Gel Electrophoresis ◽

Daughter Cell ◽

Glucose Repression ◽

Pulsed Field Gel Electrophoresis ◽

Null Mutation ◽

Electron Microscopic ◽

Transmission Electron ◽

Transmission Electron Microscopic ◽

Carboxy Terminal

An RGR1 gene product is required to repress expression of glucose-regulated genes in Saccharomyces cerevisiae. The abnormal morphology of rgr1 cells was studied. Scanning and transmission electron microscopic observations revealed that the cell wall of the daughter cell remained attached to that of mother cell. We cloned the RGR1 gene by complementation and showed that the cloned DNA was tightly linked to the chromosomal RGR1 locus. The cloned RGR1 gene suppressed all of the phenotypes caused by the mutation and encoded a 3.6-kilobase poly(A)+ RNA. The RGR1 gene is located on chromosome XII, as determined by pulsed-field gel electrophoresis, and we mapped rgr1 between gal2 and pep3 by genetic analysis. rgr1 was shown to be a new locus. We also determined the nucleotide sequence of RGR1, which was predicted to encode a 123-kilodalton protein. The null mutation resulted in lethality, indicating that the RGR1 gene is essential for growth. On the other hand, a carboxy-terminal deletion of the gene caused phenotypes similar to but more severe than those caused by the original mutation. The amount of reserve carbohydrates was reduced in rgr1 cells. Possible functions of the RGR1 product are discussed.

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Isolation and characterization of PEP3, a gene required for vacuolar biogenesis in Saccharomyces cerevisiae

Molecular and Cellular Biology ◽

10.1128/mcb.11.12.5801-5812.1991 ◽

1991 ◽

Vol 11 (12) ◽

pp. 5801-5812

Author(s):

R A Preston ◽

M F Manolson ◽

K Becherer ◽

E Weidenhammer ◽

D Kirkpatrick ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Gene Product ◽

Zinc Finger ◽

Genomic Library ◽

Sequence Similarity ◽

Carboxypeptidase Y ◽

Lag Phase ◽

Significant Similarity ◽

Reading Frame ◽

Isolation And Characterization

The Saccharomyces cerevisiae PEP3 gene was cloned from a wild-type genomic library by complementation of the carboxypeptidase Y deficiency in a pep3-12 strain. Subclone complementation results localized the PEP3 gene to a 3.8-kb DNA fragment. The DNA sequence of the fragment was determined; a 2,754-bp open reading frame predicts that the PEP3 gene product is a hydrophilic, 107-kDa protein that has no significant similarity to any known protein. The PEP3 predicted protein has a zinc finger (CX2CX13CX2C) near its C terminus that has spacing and slight sequence similarity to the adenovirus E1a zinc finger. A radiolabeled PEP3 DNA probe hybridized to an RNA transcript of 3.1 kb in extracts of log-phase and diauxic lag-phase cells. Cells bearing pep3 deletion/disruption alleles were viable, had decreased levels of protease A, protease B, and carboxypeptidase Y antigens, had decreased repressible alkaline phosphatase activity, and contained very few normal vacuolelike organelles by fluorescence microscopy and electron microscopy but had an abundance of extremely small vesicles that stained with carboxyfluorescein diacetate, were severely inhibited for growth at 37 degrees C, and were incapable of sporulating (as homozygotes). Fractionation of cells expressing a bifunctional PEP3::SUC2 fusion protein indicated that the PEP3 gene product is present at low abundance in both log-phase and stationary cells and is a vacuolar peripheral membrane protein. Sequence identity established that PEP3 and VPS18 (J. S. Robinson, T. R. Graham, and S. D. Emr, Mol. Cell. Biol. 11:5813-5824, 1991) are the same gene.

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Subcellular localization of nucleocapsid protein of SFTSV and its assembly into the ribonucleoprotein complex with L protein and viral RNA

Scientific Reports ◽

10.1038/s41598-021-01985-x ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Sithumini M. W. Lokupathirage ◽

Yoshimi Tsuda ◽

Kodai Ikegame ◽

Kisho Noda ◽

Devinda S. Muthusinghe ◽

...

Keyword(s):

Golgi Apparatus ◽

Subcellular Localization ◽

Critical Role ◽

Viral Proteins ◽

Viral Rna ◽

Host Cells ◽

N Protein ◽

L Protein ◽

Translational Activity ◽

Antiviral Targets

AbstractSevere fever with thrombocytopenia syndrome virus (SFTSV) is an emerging bunyavirus that causes novel zoonotic diseases in Asian countries including China, Japan, South Korea, and Vietnam. In phleboviruses, viral proteins play a critical role in viral particle formation inside the host cells. Viral glycoproteins (GPs) and RNA-dependent RNA polymerase (RdRp) are colocalized in the Golgi apparatus and endoplasmic reticulum-Golgi intermediate compartment (ERGIC). The nucleocapsid (N) protein was widely expressed in the cytoplasm, even in cells coexpressing GP. However, the role of SFTSV N protein remains unclear. The subcellular localization of SFTSV structural proteins was investigated using a confocal microscope. Subsequently, minigenome and immunoprecipitation assays were carried out. The N protein interacts with viral RNA (vRNA) and further shows translational activity with RdRp which is L protein and localized in the ERGIC and Golgi apparatus when co-expressed with GP. On the other hand, mutant N protein did not interact with vRNA either localized in the ERGIC or Golgi apparatus. The interaction between the N protein of SFTSV and vRNA is important for the localization of viral proteins and viral assembly. This study provides useful insights into the life cycle of SFTSV, which will lead to the detection of antiviral targets.

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Heterologous expression and characterization of Schizosaccharomyces pombe vacuolar carboxypeptidase Y in Saccharomyces cerevisiae

Current Genetics ◽

10.1007/s00294-002-0357-0 ◽

2002 ◽

Vol 42 (5) ◽

pp. 252-259 ◽

Cited By ~ 6

Author(s):

Kaoru Takegawa ◽

Sanae Tokudomi ◽

M. Shah Alam Bhuiyan ◽

Mitsuaki Tabuchi ◽

Yasuko Fujita ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Heterologous Expression ◽

Schizosaccharomyces Pombe ◽

Carboxypeptidase Y

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Genetic Interactions Between a pep7 Mutation and the PEP12 and VPS45 Genes: Evidence for a Novel SNARE Component in Transport Between the Saccharomyces cerevisiae Golgi Complex and Endosome

Genetics ◽

10.1093/genetics/147.2.467 ◽

1997 ◽

Vol 147 (2) ◽

pp. 467-478 ◽

Cited By ~ 2

Author(s):

Gene C Webb ◽

Marloes Hoedt ◽

Lynn J Poole ◽

Elizabeth W Jones

Keyword(s):

Saccharomyces Cerevisiae ◽

Golgi Complex ◽

Genetic Interactions ◽

Snare Complex ◽

Carboxypeptidase Y ◽

Fusion Reactions ◽

Vesicle Docking ◽

Transport Vesicles ◽

Allele Specific ◽

Mutant Phenotypes

The PEP7 gene from Saccharomyces cerevisiae encodes a 59-kD hydrophilic polypeptide that is required for transport of soluble vacuolar hydrolase precursors from the TGN to the endosome. This study presents the results of a high-copy suppression analysis of pep7-20 mutant phenotypes. This analysis demonstrated that both VPS45 and PEP12 are allele-specific high-copy suppressors of pep7-20 mutant phenotypes. Overexpression of VPS45 was able to completely suppress the Zn2+ sensitivity and partially suppress the carboxypeptidase Y deficiency. Overexpression of PEP12 was able to do the same, but to a lesser extent. Vps45p and Pep12p are Sec1p and syntaxin (t-SNARE) homologues, respectively, and are also thought to function in transport between the TGN and endosome. Two additional vacuole pathway SNARE complex homologues, Vps33p (Sec1p) and Pth1p (syntaxin), when overexpressed, were unable to suppress pep7-20 or any other pep7 allele, further supporting the specificity of the interactions of pep7-20 with PEP12 and VPS45. Because several other vesicle docking/fusion reactions take place in the cell without discernible participation of Pep7p homologues, we suggest that Pep7p is a step-specific regulator of docking and/or fusion of TGN-derived transport vesicles onto the endosome.

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