Localization and regulation of the cdk-activating kinase (Cak1p) from budding yeast

Eukaryotic cell cycles are controlled by the activities of cyclin-dependent kinases (cdks). The major cdk in budding yeast, Saccharomyces cerevisiae, is Cdc28p. Activation of Cdc28p requires phosphorylation on threonine 169 and binding to a cyclin. Thr-169 is phosphorylated by the cdk-activating kinase (CAK), Cak1p, which was recently identified as the physiological CAK in budding yeast. Here we present our further characterization of yeast Cak1p. We have found that Cak1p is dispersed throughout the cell as shown by immunofluorescence; biochemical subcellular fractionation confirmed that most of the Cak1p is found in the cytoplasm. Cak1p is a monomeric enzyme in crude yeast lysates. Mutagenesis of potential sites of activating phosphorylation had little effect on the activity of Cak1p in vitro or in vivo. Furthermore, Cak1p contains no posttranslational modifications detectable by two-dimensional isoelectric focusing. We found that Cak1p is a stable protein during exponential growth but that its expression decreases considerably when cells enter stationary phase. In contrast, Cak1p levels oscillate dramatically during meiosis, reflecting regulation at both the transcriptional and post-translational level. The localization and regulation of Cak1p are in contrast to those of the known vertebrate CAK, p40(MO15).

Download Full-text

Isolation and characterization of a gene (CBF2) specifying a protein component of the budding yeast kinetochore.

The Journal of Cell Biology ◽

10.1083/jcb.121.3.513 ◽

1993 ◽

Vol 121 (3) ◽

pp. 513-519 ◽

Cited By ~ 63

Author(s):

W Jiang ◽

J Lechner ◽

J Carbon

Keyword(s):

Molecular Motors ◽

Cell Biology ◽

Budding Yeast ◽

Isolation And Characterization ◽

Sequence Homologies ◽

Binding Factor ◽

Microtubule Motor

We have cloned and determined the nucleotide sequence of the gene (CBF2) specifying the large (110 kD) subunit of the 240-kD multisubunit yeast centromere binding factor CBF3, which binds selectively in vitro to yeast centromere DNA and contains a minus end-directed microtubule motor activity. The deduced amino acid sequence of CBF2p shows no sequence homologies with known molecular motors, although a consensus nucleotide binding site is present. The CBF2 gene is essential for viability of yeast and is identical to NDC10, in which a conditional mutation leads to a defect in chromosome segregation (Goh, P.-Y., and J. V. Kilmartin, in this issue of The Journal of Cell Biology). The combined in vitro and in vivo evidence indicate that CBF2p is a key component of the budding yeast kinetochore.

Download Full-text

Quantitative Characterization of a Mitotic Cyclin Threshold Regulating Exit from Mitosis

Molecular Biology of the Cell ◽

10.1091/mbc.e04-10-0897 ◽

2005 ◽

Vol 16 (5) ◽

pp. 2129-2138 ◽

Cited By ~ 32

Author(s):

Frederick R. Cross ◽

Lea Schroeder ◽

Martin Kruse ◽

Katherine C. Chen

Keyword(s):

Cell Cycle ◽

Budding Yeast ◽

Cell Cycle Control ◽

Predictive Ability ◽

Eukaryotic Cell ◽

Model Predictions ◽

Mitotic Cyclin ◽

Constitutive Overexpression

Regulation of cyclin abundance is central to eukaryotic cell cycle control. Strong overexpression of mitotic cyclins is known to lock the system in mitosis, but the quantitative behavior of the control system as this threshold is approached has only been characterized in the in vitro Xenopus extract system. Here, we quantitate the threshold for mitotic block in budding yeast caused by constitutive overexpression of the mitotic cyclin Clb2. Near this threshold, the system displays marked loss of robustness, in that loss or even heterozygosity for some regulators becomes deleterious or lethal, even though complete loss of these regulators is tolerated at normal cyclin expression levels. Recently, we presented a quantitative kinetic model of the budding yeast cell cycle. Here, we use this model to generate biochemical predictions for Clb2 levels, asynchronous as well as through the cell cycle, as the Clb2 overexpression threshold is approached. The model predictions compare well with biochemical data, even though no data of this type were available during model generation. The loss of robustness of the Clb2 overexpressing system is also predicted by the model. These results provide strong confirmation of the model's predictive ability.

Download Full-text

Yeast as a Model to Understand Actin-Mediated Cellular Functions in Mammals—Illustrated with Four Actin Cytoskeleton Proteins

Cells ◽

10.3390/cells9030672 ◽

2020 ◽

Vol 9 (3) ◽

pp. 672 ◽

Cited By ~ 1

Author(s):

Zain Akram ◽

Ishtiaq Ahmed ◽

Heike Mack ◽

Ramandeep Kaur ◽

Richard C. Silva ◽

...

Keyword(s):

Actin Cytoskeleton ◽

Molecular Mechanisms ◽

Budding Yeast ◽

Animal Cell ◽

Molecular Genetic ◽

In Vivo Studies ◽

Yeast Saccharomyces Cerevisiae ◽

Higher Eukaryotes

The budding yeast Saccharomyces cerevisiae has an actin cytoskeleton that comprises a set of protein components analogous to those found in the actin cytoskeletons of higher eukaryotes. Furthermore, the actin cytoskeletons of S. cerevisiae and of higher eukaryotes have some similar physiological roles. The genetic tractability of budding yeast and the availability of a stable haploid cell type facilitates the application of molecular genetic approaches to assign functions to the various actin cytoskeleton components. This has provided information that is in general complementary to that provided by studies of the equivalent proteins of higher eukaryotes and hence has enabled a more complete view of the role of these proteins. Several human functional homologues of yeast actin effectors are implicated in diseases. A better understanding of the molecular mechanisms underpinning the functions of these proteins is critical to develop improved therapeutic strategies. In this article we chose as examples four evolutionarily conserved proteins that associate with the actin cytoskeleton: (1) yeast Hof1p/mammalian PSTPIP1, (2) yeast Rvs167p/mammalian BIN1, (3) yeast eEF1A/eEF1A1 and eEF1A2 and (4) yeast Yih1p/mammalian IMPACT. We compare the knowledge on the functions of these actin cytoskeleton-associated proteins that has arisen from studies of their homologues in yeast with information that has been obtained from in vivo studies using live animals or in vitro studies using cultured animal cell lines.

Download Full-text

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

Biochemical Journal ◽

10.1042/bj3380403 ◽

1999 ◽

Vol 338 (2) ◽

pp. 403-407 ◽

Cited By ~ 9

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.

Download Full-text

Probing the ultrastructure of the mitotic and meiotic spindle in eggs: An expeditious approach based on semi-thin (0.25 μm) serial sections

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100076408 ◽

1983 ◽

Vol 41 ◽

pp. 552-555

Author(s):

Conly L. Rieder ◽

S. Bowser ◽

R. Nowogrodzki ◽

K. Ross ◽

G. Sluder

Keyword(s):

Small Sample Size ◽

Small Sample ◽

Meiotic Spindle ◽

Serial Sections ◽

Mitotic Apparatus ◽

Thin Sections ◽

Cell Cycles ◽

Ultrastructural Studies

Eggs have long been a favorite material for studying the mechanism of karyokinesis in-vivo and in-vitro. They can be obtained in great numbers and, when fertilized, divide synchronously over many cell cycles. However, they are not considered to be a practical system for ultrastructural studies on the mitotic apparatus (MA) for several reasons, the most obvious of which is that sectioning them is a formidable task: over 1000 ultra-thin sections need to be cut from a single 80-100 μm diameter egg and of these sections only a small percentage will contain the area or structure of interest. Thus it is difficult and time consuming to obtain reliable ultrastructural data concerning the MA of eggs; and when it is obtained it is necessarily based on a small sample size.We have recently developed a procedure which will facilitate many studies concerned with the ultrastructure of the MA in eggs. It is based on the availability of biological HVEM's and on the observation that 0.25 μm thick serial sections can be screened at high resolution for content (after mounting on slot grids and staining with uranyl and lead) by phase contrast light microscopy (LM; Figs 1-2).

Download Full-text

In vivo and in vitro characterization of immediate release dosage forms containing n-acetylcysteine using the dynamic open flow-through test apparatus

10.26226/morressier.57d6b2b7d462b8028d88dd29 ◽

2016 ◽

Author(s):

Maximilian Sager

Keyword(s):

Immediate Release ◽

Dosage Forms ◽

Test Apparatus ◽

Open Flow ◽

In Vitro Characterization ◽

Flow Through

Download Full-text

Rapid, Refined, and Robust Method for Expression, Purification, and Characterization of Recombinant Human Amyloid-beta M1-42

10.26434/chemrxiv.7725002.v1 ◽

2019 ◽

Author(s):

Priya Prakash ◽

Travis Lantz ◽

Krupal P. Jethava ◽

Gaurav Chopra

Keyword(s):

Amyloid Beta ◽

Western Blots ◽

Force Microscopy ◽

E Coli ◽

Atomic Force ◽

Set Up ◽

Short Time

Amyloid plaques found in the brains of Alzheimer’s disease (AD) patients primarily consists of amyloid beta 1-42 (Ab42). Commercially, Ab42 is synthetized using peptide synthesizers. We describe a robust methodology for expression of recombinant human Ab(M1-42) in Rosetta(DE3)pLysS and BL21(DE3)pLysS competent E. coli with refined and rapid analytical purification techniques. The peptide is isolated and purified from the transformed cells using an optimized set-up for reverse-phase HPLC protocol, using commonly available C18 columns, yielding high amounts of peptide (~15-20 mg per 1 L culture) in a short time. The recombinant Ab(M1-42) forms characteristic aggregates similar to synthetic Ab42 aggregates as verified by western blots and atomic force microscopy to warrant future biological use. Our rapid, refined, and robust technique to purify human Ab(M1-42) can be used to synthesize chemical probes for several downstream in vitro and in vivo assays to facilitate AD research.

Download Full-text

Faculty Opinions recommendation of In vitro and in vivo characterization of a novel semaphorin 3A inhibitor, SM-216289 or xanthofulvin.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1014793.196553 ◽

2003 ◽

Author(s):

Genevieve Rougon

Keyword(s):

Semaphorin 3A ◽

In Vivo Characterization

Download Full-text

Stu2p binds tubulin and undergoes an open-to-closed conformational change

The Journal of Cell Biology ◽

10.1083/jcb.200511010 ◽

2006 ◽

Vol 172 (7) ◽

pp. 1009-1022 ◽

Cited By ~ 87

Author(s):

Jawdat Al-Bassam ◽

Mark van Breugel ◽

Stephen C. Harrison ◽

Anthony Hyman

Keyword(s):

Conformational Change ◽

Conformational Transition ◽

Budding Yeast ◽

Microtubule Dynamics ◽

Open Structure ◽

Microtubule Associated Proteins ◽

Associated Proteins ◽

The Common

Stu2p from budding yeast belongs to the conserved Dis1/XMAP215 family of microtubule-associated proteins (MAPs). The common feature of proteins in this family is the presence of HEAT repeat–containing TOG domains near the NH2 terminus. We have investigated the functions of the two TOG domains of Stu2p in vivo and in vitro. Our data suggest that Stu2p regulates microtubule dynamics through two separate activities. First, Stu2p binds to a single free tubulin heterodimer through its first TOG domain. A large conformational transition in homodimeric Stu2p from an open structure to a closed one accompanies the capture of a single free tubulin heterodimer. Second, Stu2p has the capacity to associate directly with microtubule ends, at least in part, through its second TOG domain. These two properties lead to the stabilization of microtubules in vivo, perhaps by the loading of tubulin dimers at microtubule ends. We suggest that this mechanism of microtubule regulation is a conserved feature of the Dis1/XMAP215 family of MAPs.

Download Full-text