Table of contents

Volume 4, Issue 1, pp. 1 - 34, January 2017

Issue cover
Cover: Fluorescence micrograph of Schizosaccharomyces pombe cells overexpressing the copper transporter protein Ctr4 (as YFP fusion construct), which shows localization and aggregation at the cell periphery (image by Theodora Sideri and Jürg Bähler, University College London, UK); image modified by MIC. The cover is published under the Creative Commons Attribution (CC BY) license. Enlarge issue cover


Using microbes as a key tool to unravel the mechanism of autophagy and the functions of the ATG proteins

Mario Mauthe, Fulvio Reggiori

page 1-5 | 10.15698/mic2017.01.550 | Full text | PDF | Abstract

The study of microbe infections has always been a very effective approach to unveil and dissect cellular pathways. Autophagy is not an exception. Although some of the breakthrough discoveries in the field were obtained using yeast, pathogens have been and still are a great tool to discover and characterize new molecular and functional aspects of autophagy. Research on pathogens has helped to acquire knowledge about selective types of autophagy and the assembly of the autophagy machinery, i.e the autophagy-related (ATG) proteins, but also about alternative cellular roles of this pathway, such as secretion. Finally, microbes have also served to discover and characterize unconventional functions of the ATG proteins, which are uncoupled from their role in autophagy. In our recent study, we have taken advantage of viruses as a screening tool to determine the extent of the unconventional functions of the ATG proteome and characterize one of them.

Research Articles

Improvement of biochemical methods of polyP quantification

Samuel Bru, Javier Jiménez, David Canadell, Joaquín Ariño, Josep Clotet

page 6-15 | 10.15698/mic2017.01.551 | Full text | PDF | Abstract

Polyphosphate (polyP) is an abundant and physiologically important biomolecule for virtually any living cell. Therefore, determination of changes in cellular content of polyP is crucial for its functional characterization. Determination of cellular polyP has been performed by many different methods, and the lack of a standardized procedure is possibly responsible for the large dispersion of results found in the relevant literature. For a relatively simple organism, such as the yeast Saccharomyces cerevisiae, this variation can be up to 12-fold. polyP extraction and determination of free phosphate released by enzymatic degradation of the polymer is a method quite common and relatively straightforward for polyP determination. By using the yeast S. cerevisiae as model, we have experimentally evaluated the different steps in this procedure in order to identify critical issues that might explain the disparate reported results. As the main output of this evaluation we propose a straightforward and robust procedure that can be used as gold standard protocol for cellular polyP purification and determination from unicellular organisms, thus providing consistency to measurements and facilitating inter-laboratory comparisons and biological interpretation of the results.

The copper transport-associated protein Ctr4 can form prion-like epigenetic determinants in Schizosaccharomyces pombe

Theodora Sideri, Yoko Yashiroda, David A. Ellis, María Rodríguez-López, Minoru Yoshida, Mick F. Tuite, Jürg Bähler

page 16-28 | 10.15698/mic2017.01.552 | Full text | PDF | Abstract

Prions are protein-based infectious entities associated with fatal brain diseases in animals, but also modify a range of host-cell phenotypes in the budding yeast, Saccharomyces cerevisiae. Many questions remain about the evolution and biology of prions. Although several functionally distinct prion-forming proteins exist in S. cerevisiae, [HET-s] of Podospora anserina is the only other known fungal prion. Here we investigated prion-like, protein-based epigenetic transmission in the fission yeast Schizosaccharomyces pombe. We show that S. pombe cells can support the formation and maintenance of the prion form of the S. cerevisiae Sup35 translation factor [PSI+], and that the formation and propagation of these Sup35 aggregates is inhibited by guanidine hydrochloride, indicating commonalities in prion propagation machineries in these evolutionary diverged yeasts. A proteome-wide screen identified the Ctr4 copper transporter subunit as a putative prion with a predicted prion-like domain. Overexpression of the ctr4 gene resulted in large Ctr4 protein aggregates that were both detergent and proteinase-K resistant. Cells carrying such [CTR+] aggregates showed increased sensitivity to oxidative stress, and this phenotype could be transmitted to aggregate-free [ctr] cells by transformation with [CTR+] cell extracts. Moreover, this [CTR+] phenotype was inherited in a non-Mendelian manner following mating with naïve [ctr] cells, but intriguingly the [CTR+] phenotype was not eliminated by guanidine-hydrochloride treatment. Thus, Ctr4 exhibits multiple features diagnostic of other fungal prions and is the first example of a prion in fission yeast. These findings suggest that transmissible protein-based determinants of traits may be more widespread among fungi.


Toxin-mediated gene regulatory mechanism in Staphylococcus aureus

Hwang-Soo Joo and Michael Otto

page 29-31 | 10.15698/mic2017.01.553 | Full text | PDF | Abstract

The dangerous human pathogen Staphylococcus aureus relies heavily on toxins to cause disease, but toxin production can put a strong burden on the bacteria’s energy balance. Thus, controlling the synthesis of proteins solely needed in times of toxin production represents a way for the bacteria to avoid wasting energy. One hypothetical manner to accomplish this sort of regulation is by gene regulatory functions of the toxins themselves. There have been several reports about gene regulation by toxins in S. aureus, but these were never verified on the molecular level. In our study published in MBio [Joo et al., 7(5). pii: e01579-16], we show that phenol-soluble modulins (PSMs), important peptide toxins of S. aureus, release a repressor from the promoter of the operon encoding the toxin export system, thereby enabling toxin secretion. This study describes the first molecular regulatory mechanism exerted by an S. aureus toxin, setting a paradigmatic example of how S. aureus toxins may influence cell functions to adjust them to times of toxin production.

A new role for Holliday junction resolvase Yen1 in processing DNA replication intermediates exposes Dna2 as an accessory replicative helicase

Benoît Falquet, Ulrich Rass

page 32-34 | 10.15698/mic2017.01.554 | Full text | PDF | Abstract

DNA replication is mediated by a multi-protein complex known as the replisome. With the hexameric MCM (minichromosome maintenance) replicative helicase at its core, the replisome splits the parental DNA strands, forming replication forks (RFs), where it catalyses coupled leading and lagging strand DNA synthesis. While replication is a highly effective process, intrinsic and oncogene-induced replication stress impedes the progression of replisomes along chromosomes. As a consequence, RFs stall, arrest, and collapse, jeopardizing genome stability. In these instances, accessory fork progression and repair factors, orchestrated by the replication checkpoint, promote RF recovery, ensuring the chromosomes are fully replicated and can be safely segregated at cell division. Homologous recombination (HR) proteins play key roles in negotiating replication stress, binding at stalled RFs and shielding them from inappropriate processing. In addition, HR-mediated strand exchange reactions restart stalled or collapsed RFs and mediate error-free post-replicative repair. DNA transactions at stalled RFs further involve various DNA editing factors, notably helicases and nucleases. A study by Ölmezer et al. (2016) has recently identified a role for the structure-specific nuclease Yen1 (GEN1 in human) in the resolution of dead-end DNA replication intermediates after RF arrest. This new function of Yen1 is distinct from its previously known role as a Holliday junction resolvase, mediating the removal of branched HR intermediates, and it becomes essential for viable chromosome segregation in cells with a defective Dna2 helicase. These findings have revealed greater complexity in the tasks mediated by Yen1 and expose a replicative role for the elusive helicase activity of the conserved Dna2 nuclease-helicase.