Table of contents

Volume 4, Issue 6, pp. 182 - 202, June 2017

Issue cover
Cover: Live super-resolution imaging of yeast Saccharomyces cerevisiae with integrated nls-(tagRFP657)3 and Calcofluor White as markers for the nucleus and the cell wall, respectively (image by Triana Amen and Daniel Kaganovich, Hebrew University of Jerusalem, Israel); modified by MIC. The cover is published under the Creative Commons Attribution (CC BY) license. Enlarge issue cover

Research Articles

Integrative modules for efficient genome engineering in yeast

Triana Amen and Daniel Kaganovich

page 182-190 | 10.15698/mic2017.06.576 | Full text | PDF | Abstract

We present a set of vectors containing integrative modules for efficient genome integration into the commonly used selection marker loci of the yeast Saccharomyces cerevisiae. A fragment for genome integration is generated via PCR with a unique set of short primers and integrated into HIS3, URA3, ADE2, and TRP1 loci. The desired level of expression can be achieved by using constitutive (TEF1p, GPD1p), inducible (CUP1p, GAL1/10p), and daughter-specific (DSE4p) promoters available in the modules. The reduced size of the integrative module compared to conventional integrative plasmids allows efficient integration of multiple fragments. We demonstrate the efficiency of this tool by simultaneously tagging markers of the nucleus, vacuole, actin, and peroxisomes with genomically integrated fluorophores. Improved integration of our new pDK plasmid series allows stable introduction of several genes and can be used for multi-color imaging. New bidirectional promoters (TEF1p-GPD1p, TEF1p-CUP1p, and TEF1p-DSE4p) allow tractable metabolic engineering.

Research Reports

The neuroprotective steroid progesterone promotes mitochondrial uncoupling, reduces cytosolic calcium and augments stress resistance in yeast cells

Slaven Stekovic, Christoph Ruckenstuhl, Philipp Royer, Christof Winkler-Hermaden, Didac Carmona-Gutierrez, Kai-Uwe Fröhlich, Guido Kroemer, and Frank Madeo

page 191-199 | 10.15698/mic2017.06.577 | Full text | PDF | Abstract

The steroid hormone progesterone is not only a crucial sex hormone, but also serves as a neurosteroid, thus playing an important role in brain function. Epidemiological data suggest that progesterone improves the recovery of patients after traumatic brain injury. Brain injuries are often connected to elevated calcium spikes, reactive oxygen species (ROS) and programmed cell death affecting neurons. Here, we establish a yeast model to study progesterone-mediated cytoprotection. External supply of progesterone protected yeast cells from apoptosis-inducing stress stimuli and resulted in elevated mitochondrial oxygen uptake accompanied by a drop in ROS generation and ATP levels during chronological aging. In addition, cellular Ca2+ concentrations were reduced upon progesterone treatment, and this effect occurred independently of known Ca2+ transporters and mitochondrial respiration. All effects were also independent of Dap1, the yeast orthologue of the progesterone receptor. Altogether, our observations provide new insights into the cytoprotective effects of progesterone.

Microreviews

Protein aggregation triggers a declining libido in elder yeasts that still have a lust for life

Fabrice Caudron

page 200-202 | 10.15698/mic2017.06.578 | Full text | PDF | Abstract

Many organisms have to face a physiological decline that is associated with age. Humans and even budding yeast accumulate scars and cellular damages. A single yeast cell can only produce a limited number of daughter cells and thus has a finite replicative lifespan. Many studies have now identified molecular ageing factors and defects in organelle functions linked to the ageing process. However, at the cellular level, the most striking phenotype of yeast elders is their loss of mating ability. This sterility in old cells has been linked to a loss of response to mating pheromone, the peptide that haploid yeast cells send to opposite mating type cells in order to signal their presence and readiness to mate. Our results (Schlissel et al., 2017) demonstrate that old cells are unable to respond to mating pheromone due to age-induced aggregation of the protein Whi3. We recently discovered that Whi3 changes conformation and coalesces when cells experience and memorise a deceptive mating attempt. Together, these results prompt the question of how proteins physiologically aggregating behave during ageing, induce age associated phenotypes and influence the ageing process itself.