A spheroplast rate assay for determination of cell wall integrity in yeast

Ovalle, Rafael; Lim, Seung Taik; Holder, Brigitte; Jue, Chong K.; Moore, Carol W.; Lipke, Peter N.

doi:10.1002/(sici)1097-0061(19980930)14:13<1159::aid-yea317>3.0.co;2-3

Cited by 65 publications

(66 citation statements)

References 16 publications

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“…Thermotolerance was assayed as the change in cell survivorship over the course of a 2 h incubation at 48°C, estimated as CFUs on YEPD agar. Susceptibility of the yeast cell wall to zymolyase digestion was assayed by the method of Ovalle et al (92), but scaled for a 96-well format, as described in SI Materials and Methods.…”

Section: Methodsmentioning

confidence: 99%

Uncoupling reproduction from metabolism extends chronological lifespan in yeast

Nagarajan

Kruckeberg

Schmidt

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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Studies of replicative and chronological lifespan in Saccharomyces cerevisiae have advanced understanding of longevity in all eukaryotes. Chronological lifespan in this species is defined as the agedependent viability of nondividing cells. To date this parameter has only been estimated under calorie restriction, mimicked by starvation. Because postmitotic cells in higher eukaryotes often do not starve, we developed a model yeast system to study cells as they age in the absence of calorie restriction. Yeast cells were encapsulated in a matrix consisting of calcium alginate to form ∼3 mm beads that were packed into bioreactors and fed ad libitum. Under these conditions cells ceased to divide, became heat shock and zymolyase resistant, yet retained high fermentative capacity. Over the course of 17 d, immobilized yeast cells maintained >95% viability, whereas the viability of starving, freely suspended (planktonic) cells decreased to <10%. Immobilized cells exhibited a stable pattern of gene expression that differed markedly from growing or starving planktonic cells, highly expressing genes in glycolysis, cell wall remodeling, and stress resistance, but decreasing transcription of genes in the tricarboxylic acid cycle, and genes that regulate the cell cycle, including master cyclins CDC28 and CLN1. Stress resistance transcription factor MSN4 and its upstream effector RIM15 are conspicuously up-regulated in the immobilized state, and an immobilized rim15 knockout strain fails to exhibit the long-lived, growth-arrested phenotype, suggesting that altered regulation of the Rim15-mediated nutrient-sensing pathway plays an important role in extending yeast chronological lifespan under calorie-unrestricted conditions. cell longevity | Ca-alginate encapsulation U nicellular microbes senesce and die (for reviews, see refs.

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Section: Methodsmentioning

confidence: 99%

Uncoupling reproduction from metabolism extends chronological lifespan in yeast

Nagarajan

Kruckeberg

Schmidt

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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“…Spheroplast lysis was measured by following published procedures (53). BY263, BY391, BY490, and BY714 cultures were grown to log phase (OD 600 ϭ ϳ0.5) in YPD at 30°C.…”

Section: Methodsmentioning

confidence: 99%

“…The OD 600 of the cultures was checked every 5 min for each sample. Lag time, the maximal lysis rate, and the rate index were calculated as described previously (53).…”

Section: Methodsmentioning

confidence: 99%

Dissection of a Complex Phenotype by Functional Genomics Reveals Roles for the Yeast Cyclin-Dependent Protein Kinase Pho85 in Stress Adaptation and Cell Integrity

Huang

Moffat

Andrews

2002

Molecular and Cellular Biology

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Cyclin-dependent kinases (Cdks) are key regulators of the cell division cycle. Pho85 is a multifunctional Cdk in budding yeast involved in aspects of metabolism, the cell cycle, cell polarity, and gene expression. Consistent with a broad spectrum of functions, Pho85 associates with a family of 10 cyclins and deletion of PHO85 causes a pleiotropic phenotype. Discovering the physiological substrates of protein kinases is a major challenge, and we have pursued a number of genomics approaches to reveal the processes regulated by Pho85 and to understand the root cause of reduced cellular fitness in pho85⌬ mutant strains. We used a functional-genomics approach called synthetic genetic array (SGA) analysis to systematically identify strain backgrounds in which PHO85 is required for viability. In parallel, we used DNA microarrays to examine the genome-wide transcriptional consequences of deleting PHO85 or members of the Pho85 cyclin family. Using this pairwise approach coupled with phenotypic tests, we uncovered clear roles for Pho85 in cell integrity and the response to adverse growth conditions. Importantly, our combined approach allowed us to ascribe new aspects of the complex pho85 phenotype to particular cyclins; our data highlight a cell integrity function for the Pcl1,2 subgroup of Pho85 Cdks that is independent of a role for the Pho80-Pho85 kinase in the response to stress. Using a modification of the SGA technique to screen for suppressors of pho85⌬ strain growth defects, we found that deletion of putative vacuole protein gene VTC4 suppressed the sensitivity of the pho85⌬ strain to elevated CaCl 2 and many other stress conditions. Expression of VTC4 is regulated by Pho4, a transcription factor that is inhibited by the Pho80-Pho85 kinase. Genetic tests and electron microscopy experiments suggest that VTC4 is a key target of Pho4 and that Pho80-Pho85-mediated regulation of VTC4 expression is required for proper vacuole function and for yeast cell survival under a variety of suboptimal conditions. The integration of multiple genomics approaches is likely to be a generally useful strategy for extracting functional information from pleiotropic mutant phenotypes.

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“…The spheroplasts were obtained by the treatment with Zymolyase containing a protease activity with affinity for mannoproteins, and a β1,3 glucanase activity. The actions of both enzymatic activities were required to lyses yeast cell wall (Ovalle et al, 1998) and allowed us to obtain spheroplasts and the released products of the enzymatic breakdown, separately.…”

Section: Aflatoxin B 1 Releasedmentioning

confidence: 99%