Actin Cytoskeleton Regulation by the Yeast NADPH Oxidase Yno1p Impacts Processes Controlled by MAPK Pathways.

Reactive oxygen species (ROS) that exceed the antioxidative capacity of the cell can be harmful and are termed oxidative stress. Increasing evidence suggests that ROS are not exclusively detrimental, but can fulfill important signaling functions. Recently, we have been able to demonstrate that a NADPH oxidase-like enzyme (termed Yno1p) exists in the single-celled organism Saccharomyces cerevisiae. This enzyme resides in the peripheral and perinuclear endoplasmic reticulum and functions in close proximity to the plasma membrane. Its product, hydrogen peroxide, which is also produced by the action of the superoxide dismutase, Sod1p, influences signaling of key regulatory proteins Ras2p and Yck1p/2p. In the present work, we demonstrate that Yno1p-derived H2O2 regulates outputs controlled by three MAP kinase pathways that can share components: the filamentous growth (filamentous growth MAPK (fMAPK)), pheromone response, and osmotic stress response (hyperosmolarity glycerol response, HOG) pathways. A key structural component and regulator in this process is the actin cytoskeleton. The nucleation and stabilization of actin are regulated by Yno1p. Cells lacking YNO1 showed reduced invasive growth, which could be reversed by stimulation of actin nucleation. Additionally, under osmotic stress, the vacuoles of a ∆yno1 strain show an enhanced fragmentation. During pheromone response induced by the addition of alpha-factor, Yno1p is responsible for a burst of ROS. Collectively, these results broaden the roles of ROS to encompass microbial differentiation responses and stress responses controlled by MAPK pathways.

Clearing the outer mitochondrial membrane from harmful proteins via lipid droplets.

In recent years it turned out that there is not only extensive communication between the nucleus and mitochondria but also between mitochondria and lipid droplets (LDs) as well. We were able to demonstrate that a number of proteins shuttle between LDs and mitochondria and it depends on the metabolic state of the cell on which organelle these proteins are predominantly localized. Responsible for the localization of the particular proteins is a protein domain consisting of two α-helices, which we termed V-domain according to the predicted structure. So far we have detected this domain in the following proteins: mammalian BAX, BCL-XL, TCTP and yeast Mmi1p and Erg6p. According to our experiments there are two functions of this domain: (1) shuttling of proteins to mitochondria in times of stress and apoptosis; (2) clearing the outer mitochondrial membrane from pro- as well as anti-apoptotic proteins by moving them to LDs after the stress ceases. In this way the LDs are used by the cell to modulate stress response.

Formaldehyde fixation is detrimental to actin cables in glucose-depleted S. cerevisiae cells.

Actin filaments form cortical patches and emanating cables in fermenting cells of Saccharomyces cerevisiae. This pattern has been shown to be depolarized in glucose-depleted cells after formaldehyde fixation and staining with rhodamine-tagged phalloidin. Loss of actin cables in mother cells was remarkable. Here we extend our knowledge on actin in live glucose-depleted cells co-expressing the marker of actin patches (Abp1-RFP) with the marker of actin cables (Abp140-GFP). Glucose depletion resulted in appearance of actin patches also in mother cells. However, even after 80 min of glucose deprivation these cells showed a clear network of actin cables labeled with Abp140-GFP in contrast to previously published data. In live cells with a mitochondrial dysfunction (rho0 cells), glucose depletion resulted in almost immediate appearance of Abp140-GFP foci partially overlapping with Abp1-RFP patches in mother cells. Residual actin cables were clustered in patch-associated bundles. A similar overlapping "patchy" pattern of both actin markers was observed upon treatment of glucose-deprived rho+ cells with FCCP (the inhibitor of oxidative phosphorylation) and upon treatment with formaldehyde. While the formaldehyde-targeted process stays unknown, our results indicate that published data on yeast actin cytoskeleton obtained from glucose-depleted cells after fixation should be considered with caution.

Analysis of free fatty acids in beer: comparison of solid-phase extraction, solid-phase microextraction, and stir bar sorptive extraction.

Solid-phase extraction (SPE), solid-phase microextraction (SPME) using carbowax/divinylbenzen fiber, and stir bar sorptive extraction (SBSE) followed by solvent back extraction have been used for the extraction of free fatty acids (caproic, caprylic, pelargonic, capric, lauric, myristic, palmitic, stearic, oleic, linoleic, and linolenic acids) from beer. Subsequent gas chromatographic analyses with flame ionization detection were used for the determination of these compounds. Medium-chain fatty acids (caproic-lauric acid) were determined as free acids, and long-chain fatty acids (myristic-linolenic acids) were determined as methyl esters after methylation by BF(3)-methanol 14%. Linearity, recovery, and repeatability of all methods have been determined and compared with the SPE method used as a reference (SPME method was used only for medium-chain fatty acid determination). All three procedures provide similar working parameters characterized by high repeatability (2.3-16.3%) and good linearity (correlation coefficient ranging from 0.9919 to 0.9999). Results of beer analyses obtained by using these three methods were highly correlated. Although all methods provide compatible alternatives, for medium-chain fatty acid analysis SPME may be a more appropriate technique due to its operational simplicity, repeatability, and low cost.