Protein kinase A controls yeast growth in visible light.

Background: A wide variety of photosynthetic and non-photosynthetic species sense and respond to light, having developed protective mechanisms to adapt to damaging effects on DNA and proteins. While the biology of UV light-induced damage has been well studied, cellular responses to stress from visible light (400-700 nm) remain poorly understood despite being a regular part of the life cycle of many organisms. Here, we developed a high-throughput method for measuring growth under visible light stress and used it to screen for light sensitivity in the yeast gene deletion collection.

Light-sensing via hydrogen peroxide and a peroxiredoxin.

Yeast lacks dedicated photoreceptors; however, blue light still causes pronounced oscillations of the transcription factor Msn2 into and out of the nucleus. Here we show that this poorly understood phenomenon is initiated by a peroxisomal oxidase, which converts light into a hydrogen peroxide (HO) signal that is sensed by the peroxiredoxin Tsa1 and transduced to thioredoxin, to counteract PKA-dependent Msn2 phosphorylation. Upon HO, the nuclear retention of PKA catalytic subunits, which contributes to delayed Msn2 nuclear concentration, is antagonized in a Tsa1-dependent manner.

The yeast transcription factor Crz1 is activated by light in a Ca2+/calcineurin-dependent and PKA-independent manner.

Light in the visible range can be stressful to non-photosynthetic organisms. The yeast Saccharomyces cerevisiae has earlier been reported to respond to blue light via activation of the stress-regulated transcription factor Msn2p. Environmental changes also induce activation of calcineurin, a Ca(2+)/calmodulin dependent phosphatase, which in turn controls gene transcription by dephosphorylating the transcription factor Crz1p.

Continuous light exposure causes cumulative stress that affects the localization oscillation dynamics of the transcription factor Msn2p.

Light exposure is a potentially powerful stress factor during in vivo optical microscopy studies. In yeast, the general transcription factor Msn2p translocates from the cytoplasm to the nucleus in response to illumination. However, previous time-lapse fluorescence microscopy studies of Msn2p have utilized a variety of discrete exposure settings, which makes it difficult to correlate stress levels and illumination parameters.

Investigations on light-induced stress in fluorescence microscopy using nuclear localization of the transcription factor Msn2p as a reporter.

We utilized the nuclear localization of a stress-sensitive transcription factor, Msn2p, to study light-induced stress caused by time-lapse fluorescence imaging of green fluorescent protein (GFP) in budding yeast Saccharomyces cerevisiae. A range of exposure times, light intensities and intervals between exposures were tested in order to provide guidelines for noninvasive imaging. We found that the cellular response, revealed as an enhanced nuclear shuttling of Msn2p-GFP, is induced at significantly lower light exposures than those causing observable changes in cell morphology or cell growth.

Image analysis algorithms for cell contour recognition in budding yeast.

Quantification of protein abundance and subcellular localization dynamics from fluorescence microscopy images is of high contemporary interest in cell and molecular biology. For large-scale studies of cell populations and for time-lapse studies, such quantitative analysis can not be performed effectively without some kind of automated image analysis tool. Here, we present fast algorithms for automatic cell contour recognition in bright field images, optimized to the model organism budding yeast (Saccharomyces cerevisiae).

Recombinant expression, purification, and kinetic and inhibitor characterisation of human site-1-protease.

Human site-1-protease (S1P, MEROPS S08.8063), also widely known as subtilisin/kexin isozyme 1 (SKI-1), is a membrane bound subtilisin-related serine protease, that belongs to a group of nine mammalian proprotein convertases. Among these proteases, S1P displays unique substrate specificity, by showing preferred cleavage after non-basic amino acids.