Cloning and functional analysis of the dpm2 and dpm3 genes from Trichoderma reesei expressed in a Saccharomyces cerevisiae dpm1Δ mutant strain

AbstractInTrichoderma reesei, dolichyl phosphate mannose (dpm) synthase, a key enzyme in the O-glycosylation process, requires three proteins for full activity. In this study, thedpm2anddpm3genes coding for the DPMII and DPMIII subunits ofT. reeseiDPM synthase were cloned and functionally analyzed after expression in theSaccharomyces cerevisiae dpm1Δ[genotype (BY4743;his3Δ1; /leu2Δ0; lys2Δ0; /ura3Δ0; YPR183w::kanMX4] mutant. It was found that apart from the catalytic subunit DPMI, the DPMIII subunit is also essential to form an active DPM synthase in yeast. Additional expression of the DPMII protein, considered to be a regulatory subunit of DPM synthase, decreased the enzymatic activity. We also characterizedS. cerevisiaestrains expressing thedpm1,2,3ordpm1, 3genes and analyzed the consequences ofdpmexpression on protein O-glycosylationin vivoand on the cell wall composition.

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Yil102c-A is a Functional Homologue of the DPMII Subunit of Dolichyl Phosphate Mannose Synthase in Saccharomyces cerevisiae

International Journal of Molecular Sciences ◽

10.3390/ijms21238938 ◽

2020 ◽

Vol 21 (23) ◽

pp. 8938

Author(s):

Sebastian Piłsyk ◽

Urszula Perlinska-Lenart ◽

Anna Janik ◽

Elżbieta Gryz ◽

Marta Ajchler-Adamska ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Functional Analysis ◽

Trichoderma Reesei ◽

Transmembrane Domain ◽

Human Cells ◽

Catalytic Subunit ◽

Yeast Saccharomyces Cerevisiae ◽

Dolichyl Phosphate ◽

Functional Homolog ◽

Wide Range

In a wide range of organisms, dolichyl phosphate mannose (DPM) synthase is a complex of tree proteins Dpm1, Dpm2, and Dpm3. However, in the yeast Saccharomyces cerevisiae, it is believed to be a single Dpm1 protein. The function of Dpm3 is performed in S. cerevisiae by the C-terminal transmembrane domain of the catalytic subunit Dpm1. Until present, the regulatory Dpm2 protein has not been found in S. cerevisiae. In this study, we show that, in fact, the Yil102c-A protein interacts directly with Dpm1 in S. cerevisiae and influences its DPM synthase activity. Deletion of the YIL102c-A gene is lethal, and this phenotype is reversed by the dpm2 gene from Trichoderma reesei. Functional analysis of Yil102c-A revealed that it also interacts with glucosylphosphatidylinositol-N-acetylglucosaminyl transferase (GPI-GnT), similar to DPM2 in human cells. Taken together, these results show that Yil102c-A is a functional homolog of DPMII from T. reesei and DPM2 from humans.

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In vivo and in vitro studies of the purine-cytosine permease of Saccharomyces cerevisiae. Functional analysis of a mutant with an altered apparent transport constant of uptake

European Journal of Biochemistry ◽

10.1111/j.1432-1033.1992.tb16684.x ◽

1992 ◽

Vol 204 (2) ◽

pp. 699-704 ◽

Cited By ~ 17

Author(s):

Daniel BRETHES ◽

Maria-Chantal CHIRIO ◽

Christian NAPIAS ◽

Marie-Renee CHEVALLIER ◽

Jean Louis LAVIE ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Functional Analysis ◽

In Vitro Studies

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

Molecular and Cellular Biology ◽

10.1128/mcb.25.11.4359-4370.2005 ◽

2005 ◽

Vol 25 (11) ◽

pp. 4359-4370 ◽

Cited By ~ 73

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.

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Cell Surface Expression of Bacterial Esterase A by Saccharomyces cerevisiae and Its Enhancement by Constitutive Activation of the Cellular Unfolded Protein Response

Applied and Environmental Microbiology ◽

10.1128/aem.00503-06 ◽

2006 ◽

Vol 72 (11) ◽

pp. 7140-7147 ◽

Cited By ~ 24

Author(s):

Frank Breinig ◽

Björn Diehl ◽

Sabrina Rau ◽

Christian Zimmer ◽

Helmut Schwab ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Cell Wall ◽

Cell Surface ◽

Surface Display ◽

Cell Surface Display ◽

Constitutive Activation ◽

Unfolded Protein ◽

Yeast Cell Surface ◽

Protein Response

ABSTRACT Yeast cell surface display is a powerful tool for expression and immobilization of biocatalytically active proteins on a unicellular eukaryote. Here bacterial carboxylesterase EstA from Burkholderia gladioli was covalently anchored into the cell wall of Saccharomyces cerevisiae by in-frame fusion to the endogenous yeast proteins Kre1p, Cwp2p, and Flo1p. When p-nitrophenyl acetate was used as a substrate, the esterase specific activities of yeast expressing the protein fusions were 103 mU mg−1 protein for Kre1/EstA/Cwp2p and 72 mU mg−1 protein for Kre1/EstA/Flo1p. In vivo cell wall targeting was confirmed by esterase solubilization after laminarinase treatment and immunofluorescence microscopy. EstA expression resulted in cell wall-associated esterase activities of 2.72 U mg−1 protein for Kre1/EstA/Cwp2p and 1.27 U mg−1 protein for Kre1/EstA/Flo1p. Furthermore, esterase display on the yeast cell surface enabled the cells to effectively grow on the esterase-dependent carbon source glycerol triacetate (Triacetin). In the case of Kre1/EstA/Flo1p, in vivo maturation within the yeast secretory pathway and final incorporation into the wall were further enhanced when there was constitutive activation of the unfolded protein response pathway. Our results demonstrate that esterase cell surface display in yeast, which, as shown here, is remarkably more effective than EstA surface display in Escherichia coli, can be further optimized by activating the protein folding machinery in the eukaryotic secretion pathway.

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Nuclear congression is driven by cytoplasmic microtubule plus end interactions in S. cerevisiae

The Journal of Cell Biology ◽

10.1083/jcb.200510032 ◽

2005 ◽

Vol 172 (1) ◽

pp. 27-39 ◽

Cited By ~ 37

Author(s):

Jeffrey N. Molk ◽

E.D. Salmon ◽

Kerry Bloom

Keyword(s):

Saccharomyces Cerevisiae ◽

Cell Wall ◽

Binding Proteins ◽

Budding Yeast ◽

Model System ◽

Organelle Transport ◽

Nuclear Movement ◽

Cytoplasmic Microtubule ◽

Cell Wall Breakdown

Nuclear movement before karyogamy in eukaryotes is known as pronuclear migration or as nuclear congression in Saccharomyces cerevisiae. In this study, S. cerevisiae is used as a model system to study microtubule (MT)-dependent nuclear movements during mating. We find that nuclear congression occurs through the interaction of MT plus ends rather than sliding and extensive MT overlap. Furthermore, the orientation and attachment of MTs to the shmoo tip before cell wall breakdown is not required for nuclear congression. The MT plus end–binding proteins Kar3p, a class 14 COOH-terminal kinesin, and Bik1p, the CLIP-170 orthologue, localize to plus ends in the shmoo tip and initiate MT interactions and depolymerization after cell wall breakdown. These data support a model in which nuclear congression in budding yeast occurs by plus end MT capture and depolymerization, generating forces sufficient to move nuclei through the cytoplasm. This is the first evidence that MT plus end interactions from oppositely oriented organizing centers can provide the force for organelle transport in vivo.

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Resistance to the plant PR-5 protein osmotin in the model fungus Saccharomyces cerevisiae is mediated by the regulatory effects of SSD1 on cell wall composition

The Plant Journal ◽

10.1046/j.1365-313x.2001.00967.x ◽

2001 ◽

Vol 25 (3) ◽

pp. 271-280 ◽

Cited By ~ 40

Author(s):

José Ignacio Ibeas ◽

Dae-Jin Yun ◽

Barbara Damsz ◽

Meena L. Narasimhan ◽

Yukifumi Uesono ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Cell Wall ◽

Cell Wall Composition ◽

Regulatory Effects

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Functional relationships between the Saccharomyces cerevisiae cis-prenyltransferases required for dolichol biosynthesis.

Acta Biochimica Polonica ◽

10.18388/abp.2005_3511 ◽

2005 ◽

Vol 52 (1) ◽

pp. 221-232 ◽

Cited By ~ 10

Author(s):

Kariona Grabińska ◽

Grazyna Sosińska ◽

Jacek Orłowski ◽

Ewa Swiezewska ◽

Thierry Berges ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Enzymatic Activity ◽

Farnesyl Diphosphate ◽

Farnesyl Diphosphate Synthase ◽

Gene Encoding ◽

Yeast Saccharomyces Cerevisiae ◽

Functional Relationships ◽

Dolichyl Phosphate ◽

Preferential Synthesis

In the yeast Saccharomyces cerevisiae the RER2 and SRT1 genes encode Rer2 and Srt1 proteins with cis-prenyltransferase (cis-PT-ase) activity. Both cis-PT-ases utilize farnesyl diphosphate (FPP) as a starter for polyprenyl diphosphate (dolichol backbone) formation. The products of the Rer2 and Srt1 proteins consist of 14-17 and 18-23 isoprene units, respectively. In this work we demonstrate that deletion or overexpression of SRT1 up-regulates the activity of Rer2p and dolichol content. However, upon overexpression of SRT1, preferential synthesis of longer-chain dolichols and a decrease in the amount of the shorter species are observed. Furthermore, overexpression of the ERG20 gene (encoding farnesyl diphosphate synthase, Erg20p) induces transcription of SRT1 mRNA and increases the levels of mRNA for RER2 and DPM1 (dolichyl phosphate mannose synthase, Dpm1p). Subsequently the enzymatic activity of Rer2p and dolichol content are also increased. However, the amount of Dpm1p or its enzymatic activity remain unchanged.

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Independent evolution of polymerization in the Actin ATPase clan regulates hexokinase activity

10.1101/686915 ◽

2019 ◽

Cited By ~ 2

Author(s):

Patrick R Stoddard ◽

Eric M. Lynch ◽

Daniel P. Farrell ◽

Quincey A. Justman ◽

Annie M. Dosey ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Enzymatic Activity ◽

Maximum Rate ◽

Protein Fold ◽

Hexokinase Activity ◽

Sugar Addition ◽

Glucose Phosphorylation ◽

Ascomycete Fungi ◽

Actin Protein

AbstractThe actin protein fold is found in cytoskeletal polymers, chaperones, and various metabolic enzymes. Many actin-fold proteins, like the carbohydrate kinases, do not polymerize. We find that Glk1, aSaccharomyces cerevisiaeglucokinase, forms two-stranded filaments with unique ultrastructure, distinct from that of cytoskeletal polymers. In cells, Glk1 polymerizes upon sugar addition and depolymerizes upon sugar withdrawal. Glk1 polymerization inhibits its enzymatic activity, thus the Glk1 monomer-polymer equilibrium sets a maximum rate of glucose phosphorylation regardless of Glk1 concentration. A mutation eliminating Glk1 polymerization alleviates concentration-dependent enzyme inhibition, causing glucokinase activity to become unconstrained. Polymerization-based regulation of Glk1 activity serves an important functionin vivo: yeast containing non-polymerizing Glk1 are less fit when growing on sugars and more likely to die when refed glucose. Glucokinase polymerization arose within the ascomycete fungi and is conserved across a group of divergent (150-200 mya) yeast. We show that Glk1 polymerization arose independently from other actin-related filaments and allows yeast to rapidly modulate glucokinase activity as nutrient availability changes.One-sentence summaryYeast glucokinase activity is limited by its polymerization, which is critical for cell viability during glucose refeeding.

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Adenovirus Protein E4orf4 Induces Premature APCCdc20 Activation in Saccharomyces cerevisiae by a Protein Phosphatase 2A-Dependent Mechanism

Journal of Virology ◽

10.1128/jvi.02434-09 ◽

2010 ◽

Vol 84 (9) ◽

pp. 4798-4809 ◽

Cited By ~ 25

Author(s):

Melissa Z. Mui ◽

Diana E. Roopchand ◽

Matthew S. Gentry ◽

Richard L. Hallberg ◽

Jackie Vogel ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Protein Phosphatase ◽

Protein Phosphatase 2A ◽

Functional Characterization ◽

Specific Inhibitor ◽

Human Adenovirus ◽

Yeast Cells ◽

Regulatory Subunit ◽

Phosphatase 2A

ABSTRACT Protein phosphatase 2A (PP2A) has been implicated in cell cycle progression and mitosis; however, the complexity of PP2A regulation via multiple B subunits makes its functional characterization a significant challenge. The human adenovirus protein E4orf4 has been found to induce both high Cdk1 activity and the accumulation of cells in G2/M in both mammalian and yeast cells, effects which are largely dependent on the B55/Cdc55 regulatory subunit of PP2A. Thus, E4orf4 represents a unique means by which the function of a specific form of PP2A can be delineated in vivo. In Saccharomyces cerevisiae, only two PP2A regulatory subunits exist, Cdc55 and Rts1. Here, we show that E4orf4-induced toxicity depends on a functional interaction with Cdc55. E4orf4 expression correlates with the inappropriate reduction of Pds1 and Scc1 in S-phase-arrested cells. The unscheduled loss of these proteins suggests the involvement of PP2ACdc55 in the regulation of the Cdc20 form of the anaphase-promoting complex (APC). Contrastingly, activity of the Hct1 form of the APC is not induced by E4orf4, as demonstrated by the observed stability of its substrates. We propose that E4orf4, being a Cdc55-specific inhibitor of PP2A, demonstrates the role of PP2ACdc55 in regulating APCCdc20 activity.

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