scholarly journals Genetic and Biochemical Characterization of the Yeast Spo12 Protein

1999 ◽  
Vol 10 (11) ◽  
pp. 3689-3703 ◽  
Author(s):  
Megan E. Grether ◽  
Ira Herskowitz
Keyword(s):  
Biochemical Analysis ◽  
Biochemical Characterization ◽  
Nuclear Division ◽  
Budding Yeast ◽  
Hybrid Analysis ◽  
Vegetative Cells ◽  
Synthetically Lethal ◽  
Cdc28 Kinase ◽  
Two Hybrid

We have performed a genetic and biochemical analysis of theSPO12 gene, which regulates meiotic nuclear divisions in budding yeast. When sporulated, spo12 mutants undergo a single meiotic nuclear division most closely resembling meiosis II. We observed that Spo12 protein is localized to the nucleus during both meiotic divisions and that Clb1-Cdc28, Clb3-Cdc28, Clb4-Cdc28, and Clb5-Cdc28 kinase activities during meiosis were not affected by a spo12 mutation. Using two-hybrid analysis, we identified several genes, three of which are meiotically induced, that may code for proteins that interact with Spo12p. We also observed that two genes, BNS1 (Bypasses Need for Spo12p), which has homology to SPO12, andSPO13, whose mutant phenotype is like that ofspo12, can partially suppress the meiotic defect ofspo12 mutants when overexpressed. We found that Spo12p is also localized to the nucleus in vegetative cells and that its level peaks during G2/M. We observed that a spo12 mutation is synthetically lethal in vegetative cells with a mutation inHCT1, a gene necessary for cells to exit mitosis, suggesting that Spo12p may have a role in exit from mitosis.

scholarly journals The Human TFIID Components TAFII135 and TAFII20 and the Yeast SAGA Components ADA1 and TAFII68 Heterodimerize to Form Histone-Like Pairs

2000 ◽  
Vol 20 (1) ◽  
pp. 340-351 ◽  
Author(s):  
Yann-Gaël Gangloff ◽  
Sebastiaan Werten ◽  
Christophe Romier ◽  
Lucie Carré ◽  
Olivier Poch ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Saccharomyces Cerevisiae ◽  
Biochemical Characterization ◽  
Hybrid Analysis ◽  
Histone Fold ◽  
Histone Octamer ◽  
Fold Type ◽  
Transcription Complexes ◽  
Two Hybrid

ABSTRACT It has been previously proposed that the transcription complexes TFIID and SAGA comprise a histone octamer-like substructure formed from a heterotetramer of H4-like human hTAFII80 (or itsDrosophila melanogaster dTAFII60 and yeast [Saccharomyces cerevisiae] yTAFII60 homologues) and H3-like hTAFII31 (dTAFII40 and yTAFII17) along with two homodimers of H2B-like hTAFII20 (dTAFII30α and yTAFII61/68). However, it has not been formally shown that hTAFII20 heterodimerizes via its histone fold. By two-hybrid analysis with yeast and biochemical characterization of complexes formed by coexpression in Escherichia coli, we showed that hTAFII20 does not homodimerize but heterodimerizes with hTAFII135. Heterodimerization requires the α2 and α3 helices of the hTAFII20 histone fold and is abolished by mutations in the hydrophobic face of the hTAFII20 α2 helix. Interaction with hTAFII20 requires a domain of hTAFII135 which shows sequence homology to H2A. This domain also shows homology to the yeast SAGA component ADA1, and we show that yADA1 heterodimerizes with the histone fold region of yTAFII61/68, the yeast hTAFII20 homologue. These results are indicative of a histone fold type of interaction between hTAFII20-hTAFII135 and yTAFII68-yADA1, which therefore constitute novel histone-like pairs in the TFIID and SAGA complexes.

scholarly journals Characterization of the interaction domains of Ure2p, a prion-like protein of yeast

1999 ◽  
Vol 338 (2) ◽  
pp. 403-407 ◽  
Author(s):  
Eric FERNANDEZ-BELLOT ◽  
Elisabeth GUILLEMET ◽  
Agnès BAUDIN-BAILLIEU ◽  
Sébastien GAUMER ◽  
Anton A. KOMAR ◽  
...  
Keyword(s):  
Catalytic Domain ◽  
Deletion Mutants ◽  
Loss Of Function ◽  
Hybrid Analysis ◽  
Yeast Saccharomyces Cerevisiae ◽  
Interaction Domains ◽  
Two Hybrid

In the yeast Saccharomyces cerevisiae, the non-Mendelian inherited genetic element [URE3] behaves as a prion. A hypothesis has been put forward which states that [URE3] arises spontaneously from its cellular isoform Ure2p (the product of the URE2 gene), and propagates through interactions of the N-terminal domain of the protein, thus leading to its aggregation and loss of function. In the present study, various N- and C-terminal deletion mutants of Ure2p were constructed and their cross-interactions were tested in vitro and in vivo using affinity binding and a two-hybrid analysis. We show that the self-interaction of the protein is mediated by at least two domains, corresponding to the first third of the protein (the so-called prion-forming domain) and the C-terminal catalytic domain.

Pep3p/Pep5p Complex: A Putative Docking Factor at Multiple Steps of Vesicular Transport to the Vacuole of Saccharomyces cerevisiae

Genetics ◽  
2000 ◽  
Vol 156 (1) ◽  
pp. 105-122 ◽  
Author(s):  
Amit Srivastava ◽  
Carol A Woolford ◽  
Elizabeth W Jones
Keyword(s):  
Vacuolar Membrane ◽  
Snare Complex ◽  
Snare Proteins ◽  
Fusion Reactions ◽  
Hybrid Analysis ◽  
Transport Pathways ◽  
Transport Vesicles ◽  
Oligomeric Complex ◽  
Two Hybrid

Abstract Pep3p and Pep5p are known to be necessary for trafficking of hydrolase precursors to the vacuole and for vacuolar biogenesis. These proteins are present in a hetero-oligomeric complex that mediates transport at the vacuolar membrane. PEP5 interacts genetically with VPS8, implicating Pep5p in the earlier Golgi to endosome step and/or in recycling from the endosome to the Golgi. To understand further the cellular roles of Pep3p and Pep5p, we isolated and characterized a set of pep3 conditional mutants. Characterization of mutants revealed that pep3ts mutants are defective in the endosomal and nonendosomal Golgi to vacuole transport pathways, in the cytoplasm to vacuole targeting pathway, in recycling from the endosome back to the late Golgi, and in endocytosis. PEP3 interacts genetically with two members of the endosomal SNARE complex, PEP12 (t-SNARE) and PEP7 (homologue of mammalian EEA1); Pep3p and Pep5p associate physically with Pep7p as revealed by two-hybrid analysis. Our results suggest that a core Pep3p/Pep5p complex promotes vesicular docking/fusion reactions in conjunction with SNARE proteins at multiple steps in transport routes to the vacuole. We propose that this complex may be responsible for tethering transport vesicles on target membranes.

scholarly journals Biochemical and functional characterization of a meiosis-specific Pch2/ORC AAA+ assembly

2020 ◽  
Vol 3 (11) ◽  
pp. e201900630
Author(s):  
María Ascensión Villar-Fernández ◽  
Richard Cardoso da Silva ◽  
Magdalena Firlej ◽  
Dongqing Pan ◽  
Elisabeth Weir ◽  
...  
Keyword(s):  
Biochemical Analysis ◽  
Budding Yeast ◽  
Functional Characterization ◽  
Efficient Removal ◽  
Terminal Domain ◽  
Origins Of Replication ◽  
Mcm Helicase ◽  
A Subunit ◽  
Aaa Protein

Pch2 is a meiosis-specific AAA+ protein that controls several important chromosomal processes. We previously demonstrated that Orc1, a subunit of the ORC, functionally interacts with budding yeast Pch2. The ORC (Orc1-6) AAA+ complex loads the AAA+ MCM helicase to origins of replication, but whether and how ORC collaborates with Pch2 remains unclear. Here, we show that a Pch2 hexamer directly associates with ORC during the meiotic G2/prophase. Biochemical analysis suggests that Pch2 uses its non-enzymatic NH2-terminal domain and AAA+ core and likely engages the interface of ORC that also binds to Cdc6, a factor crucial for ORC-MCM binding. Canonical ORC function requires association with origins, but we show here that despite causing efficient removal of Orc1 from origins, nuclear depletion of Orc2 and Orc5 does not trigger Pch2/Orc1-like meiotic phenotypes. This suggests that the function for Orc1/Pch2 in meiosis can be executed without efficient association of ORC with origins of replication. In conclusion, we uncover distinct functionalities for Orc1/ORC that drive the establishment of a non-canonical, meiosis-specific AAA+ assembly with Pch2.

scholarly journals Biochemical Characterization of Gyp6p, a Ypt/Rab-specific GTPase-activating Protein from Yeast

2001 ◽  
Vol 276 (15) ◽  
pp. 12135-12139 ◽  
Author(s):  
Elke Will ◽  
Dieter Gallwitz
Keyword(s):  
Biochemical Characterization ◽  
Three Dimensional ◽  
Significant Inhibition ◽  
Dimensional Structure ◽  
Physical Interaction ◽  
Mutant Proteins ◽  
Gtpase Activating Proteins ◽  
Two Hybrid ◽  
Intrinsic Gtpase Activity

Gyp6p from yeast belongs to the GYP family of Ypt/Rab-specific GTPase-activating proteins, and Ypt6p is its preferred substrate (Strom, M., Vollmer, P., Tan, T. J., and Gallwitz, D. (1993)Nature361, 736–739). We have investigated the kinetic parameters of Gyp6p/Ypt6p interactions and find that Gyp6p accelerates the intrinsic GTPase activity of Ypt6p (0.0002 min−1) by a factor of 5 × 106and that they have a very low affinity for its preferred substrate(Km= 592 μm). Substitution with alanine of several arginines, which Gyp6p shares with other GYP family members, resulted in significant inhibition of GAP activity. Replacement of arginine-155 with either alanine or lysine abolished its GAP activity, indicating a direct involvement of this strictly conserved arginine in catalysis. Physical interaction of the catalytically inactive Gyp6(R155A) mutant GAP with Ypt6 wild-type and Ypt6 mutant proteins could be demonstrated with the two-hybrid system. Short N-terminal and C-terminal truncations of Gyp6p resulted in a complete loss of GAP activity and Ypt6p binding, showing that in contrast to two other Gyp proteins studied previously, most of the 458 amino acid-long Gyp6p sequence is required to form a three-dimensional structure that allows substrate binding and catalysis.

scholarly journals Morphological, Functional and Biochemical Characterization of Canine Gingival Fibroblasts

2013 ◽  
Vol 24 (2) ◽  
pp. 128-135 ◽  
Author(s):  
Camila Bonvicino Pelegrini ◽  
Luciana Prado Maia ◽  
Sérgio Luís Scombatti de Souza ◽  
Mário Taba Jr ◽  
Daniela Bazan Palioto
Keyword(s):  
Protein Content ◽  
Total Protein ◽  
Biochemical Analysis ◽  
Biochemical Characterization ◽  
In Vivo Studies ◽  
Total Protein Content ◽  
Gingival Fibroblasts

As dogs are good models for in vivo studies, it is interesting to evaluate the behavior of canine gingival fibroblasts (CGF) in vitro, so that these cells could be seeded on a matrix and later studied in vivo. The aim of this study was to perform a morphological, functional and biochemical analysis of CGF, comparing it with human gingival fibroblasts (HGF), as well as to evaluate the change of their characteristics over several passages. Using gingival fibroblasts from 3 dogs and 3 humans in the subculture (Sub), first (P1), third (P3), fifth (P5) and seventh (P7) passages, the following parameters were assessed: cell morphology, spreading, adhesion, viability and total protein content. The results showed no major differences between the passages in terms of morphology and spreading, and a tendency of greater adhesion and viability for HGF when compared with CGF. The total protein content was significantly higher for HGF. HGF exhibited greater functional and biochemical activity in vitro compared to CGF. Higher numbers at Sub were observed for both CGF and HGF in all evaluated parameters. The differences do not prevent the use of CGF for tissue engineering, but its use seems to be more appropriate in the subculture or first passage.

scholarly journals Atg17 Functions in Cooperation with Atg1 and Atg13 in Yeast Autophagy

2005 ◽  
Vol 16 (5) ◽  
pp. 2544-2553 ◽  
Author(s):  
Yukiko Kabeya ◽  
Yoshiaki Kamada ◽  
Misuzu Baba ◽  
Hirosato Takikawa ◽  
Mitsuru Sasaki ◽  
...  
Keyword(s):  
Kinase Activity ◽  
Biochemical Characterization ◽  
Eukaryotic Cells ◽  
Autophagosome Formation ◽  
Yeast Saccharomyces Cerevisiae ◽  
Starvation Condition ◽  
Mutant Cells ◽  
Two Hybrid ◽  
Starvation Conditions

In eukaryotic cells, nutrient starvation induces the bulk degradation of cellular materials; this process is called autophagy. In the yeast Saccharomyces cerevisiae, most of the ATG (autophagy) genes are involved in not only the process of degradative autophagy, but also a biosynthetic process, the cytoplasm to vacuole (Cvt) pathway. In contrast, the ATG17 gene is required specifically in autophagy. To better understand the function of Atg17, we have performed a biochemical characterization of the Atg17 protein. We found that the atg17Δ mutant under starvation condition was largely impaired in autophagosome formation and only rarely contained small autophagosomes, whose size was less than one-half of normal autophagosomes in diameter. Two-hybrid analyses and coimmunoprecipitation experiments demonstrated that Atg17 physically associates with Atg1-Atg13 complex, and this binding was enhanced under starvation conditions. Atg17-Atg1 binding was not detected in atg13Δ mutant cells, suggesting that Atg17 interacts with Atg1 through Atg13. A point mutant of Atg17, Atg17C24R, showed reduced affinity for Atg13, resulting in impaired Atg1 kinase activity and significant defects in autophagy. Taken together, these results indicate that Atg17-Atg13 complex formation plays an important role in normal autophagosome formation via binding to and activating the Atg1 kinase.

scholarly journals Cdc5 Interacts with the Wee1 Kinase in Budding Yeast

2001 ◽  
Vol 21 (15) ◽  
pp. 4949-4959 ◽  
Author(s):  
Clinton R. Bartholomew ◽  
Sung Ho Woo ◽  
Yun Shin Chung ◽  
Carolyn Jones ◽  
Christopher F. J. Hardy
Keyword(s):  
Budding Yeast ◽  
Affinity Purification ◽  
Negative Regulator ◽  
Hybrid Analysis ◽  
Bud Formation ◽  
Multinucleate Cells ◽  
Inactive Form ◽  
Unicellular Organisms ◽  
Wee1 Kinase ◽  
Two Hybrid

ABSTRACT Development of a multicellular organism requires that mitosis and morphogenesis be coordinated. These processes must also be synchronized during the growth of unicellular organisms. In the yeastSaccharomyces cerevisiae, mitosis is dependent on the prior growth of a daughter cell in the form of a bud. Overexpression of wild-type Polo-like kinase Cdc5 or a catalytically inactive form resulted in the formation of multinucleate cells in budding yeast. Immunofluorescence analysis of these multinulceate cells showed that mitosis and bud formation were no longer linked. Others have shown that Swe1 is required for coupling mitosis to bud formation during a perturbed cell cycle. When the normal pathway of bud formation is perturbed, Swe1 functions to delay mitosis through negative regulation of Clb/Cdk. In cells lacking Swe1, multinucleate cells are formed in response to delays in bud formation. Affinity purification, two-hybrid analysis, and mutant characterization results suggested that Cdc5 and Swe1 interact. From these results, we conclude that multinucleate formation in response to Cdc5 overexpression is linked to titration of Swe1 function. These results also suggest that Cdc5 may be a negative regulator of Swe1.

Purification and biochemical characterization of tubulin from the budding yeast Saccharomyces cerevisiae

Biochemistry ◽  
1993 ◽  
Vol 32 (34) ◽  
pp. 8823-8835 ◽  
Author(s):  
Ashley Davis ◽  
Carleton R. Sage ◽  
Leslie Wilson ◽  
Kevin W. Farrell
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Biochemical Characterization ◽  
Budding Yeast ◽  
Yeast Saccharomyces Cerevisiae

scholarly journals Development of Tools for the Biochemical Characterization of the Symbiotic Receptor-Like Kinase DMI2

2013 ◽  
Vol 26 (2) ◽  
pp. 216-226 ◽  
Author(s):  
Brendan K. Riely ◽  
Estíbaliz Larrainzar ◽  
Cara H. Haney ◽  
Jeong-Hwan Mun ◽  
Erena Gil-Quintana ◽  
...  
Keyword(s):  
Biochemical Analysis ◽  
Biochemical Characterization ◽  
Expression Patterns ◽  
Affinity Tag ◽  
High Molecular Weight Complex ◽  
Nod Factor ◽  
Cytoplasmic Vesicles ◽  
Receptor Like Kinase

The Medicago truncatula DMI2 gene encodes a leucine-rich repeat receptor-like kinase that is essential for symbiosis with nitrogen-fixing rhizobia. While phenotypic analyses have provided a description for the host's responses mediated by DMI2, a lack of tools for in vivo biochemical analysis has hampered efforts to elucidate the mechanisms by which DMI2 mediates symbiotic signal transduction. Here, we report stably transformed M. truncatula lines that express a genomic DMI2 construct that is fused to a dual-affinity tag containing three copies of the hemagglutinin epitope and a single StrepII tag (gDMI2:HAST). gDMI2: HAST complements the dmi2-1 mutation, and transgenic plants expressing this construct behave similarly to wild-type plants. We show that the expression patterns of gDMI2:HAST recapitulate those of endogenous DMI2 and that we can detect and purify DMI2:HAST from microsomal root and nodule extracts. Using this line, we show that DMI2 resides in a high–molecular weight complex, which is consistent with our observation that DMI2:GFP localizes to plasma membrane–associated puncta and cytoplasmic vesicles. We further demonstrate that Nod factor (NF) perception increases the abundance of DMI2 vesicles. These tools should be a valuable resource for the Medicago community to dissect the biochemical function of DMI2.

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