Microbiology

In this protocol, we describe the analysis of protein stability over time, using synthesis shutoff. As an example, we express HA-tagged yeast mitofusin Fzo1 in Saccharomyces cerevisiae and inhibit translation via cycloheximide (CHX). Proteasomal inhibition with MG132 is performed, as an optional step, before the addition of CHX. Proteins are extracted via trichloroacetic acid (TCA) precipitation and subsequently separated via SDS-PAGE. Immunoblotting and antibody-decoration are performed to detect Fzo1 using HA-specific antibodies. We have adapted the method of blocking protein translation with cycloheximide to analyze the stability of high molecular weight proteins, including post-translational modifications and their impact on protein turnover.

Yeasts have provided an exceptional model for studying metabolism and bioenergetics in eukaryotic cells. Among numerous metabolites, adenosine triphosphate (ATP) is a major metabolite that is essential for all living organisms. Therefore, a clearer understanding of ATP dynamics in living yeast cells is important for deciphering cellular energy metabolism. However, none of the methods currently available to measure ATP, including biochemical analyses and ATP indicators, have been suitable for close examinations of ATP concentrations in yeast cells at the single cell level. Using the recently developed ATP biosensor QUEEN, which is suitable for yeasts and bacteria, a protocol was described herein to visualize ATP concentrations in living budding and fission yeast cells. This simple method enables the easy and reliable examination of ATP dynamics in various yeast mutants, thereby providing novel molecular insights into cellular energy metabolism.

The yeast Saccharomyces cerevisiae (S. cerevisiae) harboring ade1 or ade2 mutations manifest red colony color phenotype on rich yeast medium YPD. In these mutants, intermediate metabolites of adenine biosynthesis pathway are accumulated. Accumulated intermediates, in the presence of reduced glutathione, are transported to the vacuoles, whereupon the development of the red color phenotype occurs. Here, we describe a method to score for presence of oxidative stress upon expression of amyloid-like proteins that would convert the red phenotype of ade1/ade2 mutant yeast to white. This assay could be a useful tool for screening for drugs with anti-amyloid aggregation or anti-oxidative stress potency.

Aspergillus fumigatus is a ubiquitous fungal pathogen that forms airborne conidia. The process of restricting conidial germination into hyphae by lung leukocytes is critical in determining infectious outcomes. Tracking the outcome of conidia-host cell encounters in vivo is technically challenging and an obstacle to understanding the molecular and cellular basis of antifungal immunity in the lung. Here, we describe a method that utilizes a genetically engineered Aspergillus strain [called FLARE (Jhingran et al., 2012; Espinosa et al., 2014; Heung et al., 2015)] to monitor conidial phagocytosis and killing by leukocytes within the lung environment at single encounter resolution.