Distinct redox regulation in sub-cellular compartments in response to various stress conditions in Saccharomyces cerevisiae

Responses to many growth and stress conditions are assumed to act via changes to the cellular redox status. However, direct measurement of pH-adjusted redox state during growth and stress has never been carried out. Organellar redox state (EGSH) was measured using the fluorescent probes roGFP2 and pHluorin in Saccharomyces cerevisiae. In particular, we investigated changes in organellar redox state in response to various growth and stress conditions to better understand the relationship between redox-, oxidative- and environmental stress response systems. EGSH values of the cytosol, mitochondrial matrix and peroxisome were determined in exponential and stationary phase in various media. These values (-340 to -350 mV) were more reducing than previously reported. Interestingly, sub-cellular redox state remained unchanged when cells were challenged with stresses previously reported to affect redox homeostasis. Only hydrogen peroxide and heat stress significantly altered organellar redox state. Hydrogen peroxide stress altered the redox state of the glutathione disulfide/glutathione couple (GSSG, 2H+/2GSH) and pH. Recovery from moderate hydrogen peroxide stress was most rapid in the cytosol, followed by the mitochondrial matrix, with the peroxisome the least able to recover. Conversely, the bulk of the redox shift observed during heat stress resulted from alterations in pH and not the GSSG, 2H+/2GSH couple. This study presents the first direct measurement of pH-adjusted redox state in sub-cellular compartments during growth and stress conditions. Redox state is distinctly regulated in organelles and data presented challenge the notion that perturbation of redox state is central in the response to many stress conditions

The GABARAP Co-Secretome Identified by APEX2-GABARAP Proximity Labelling of Extracellular Vesicles

The autophagy-related ATG8 protein GABARAP has not only been shown to be involved in the cellular self-degradation process called autophagy but also fulfils functions in intracellular trafficking processes such as receptor transport to the plasma membrane. Notably, available mass spectrometry data suggest that GABARAP is also secreted into extracellular vesicles (EVs). Here, we confirm this finding by the immunoblotting of EVs isolated from cell culture supernatants and human blood serum using specific anti-GABARAP antibodies. To investigate the mechanism by which GABARAP is secreted, we applied proximity labelling, a method for studying the direct environment of a protein of interest in a confined cellular compartment. By expressing an engineered peroxidase (APEX2)-tagged variant of GABARAP—which, like endogenous GABARAP, was present in EVs prepared from HEK293 cells—we demonstrate the applicability of APEX2-based proximity labelling to EVs. The biotinylated protein pool which contains the APEX2-GABARAP co-secretome contained not only known GABARAP interaction partners but also proteins that were found in APEX2-GABARAP’s proximity inside of autophagosomes in an independent study. All in all, we not only introduce a versatile tool for co-secretome analysis in general but also uncover the first details about autophagy-based pathways as possible biogenesis mechanisms of GABARAP-containing EVs

Lack of GABARAP-Type Proteins Is Accompanied by Altered Golgi Morphology and Surfaceome Composition

GABARAP (γ-aminobutyric acid type A receptor-associated protein) and its paralogues GABARAPL1 and GABARAPL2 comprise a subfamily of autophagy-related Atg8 proteins. They are studied extensively regarding their roles during autophagy. Originally, however, especially GABARAPL2 was discovered to be involved in intra-Golgi transport and homotypic fusion of post-mitotic Golgi fragments. Recently, a broader function of mammalian Atg8s on membrane trafficking through interaction with various soluble N-ethylmaleimide-sensitive factor-attachment protein receptors SNAREs was suggested. By immunostaining and microscopic analysis of the Golgi network, we demonstrate the importance of the presence of individual GABARAP-type proteins on Golgi morphology. Furthermore, triple knockout (TKO) cells lacking the whole GABARAP subfamily showed impaired Golgi-dependent vesicular trafficking as assessed by imaging of fluorescently labelled ceramide. With the Golgi apparatus being central within the secretory pathway, we sought to investigate the role of the GABARAP-type proteins for cell surface protein trafficking. By analysing the surfaceome compositionofTKOs, we identified a subset of cell surface proteins with altered plasma membrane localisation. Taken together, we provide novel insights into an underrated aspect of autophagy-independent functions of the GABARAP subfamily and recommend considering the potential impact of GABARAP subfamily proteins on a plethora of processes during experimental analysis of GABARAP-deficient cells not only in the autophagic context

Autophagy-Related Proteins GABARAP and LC3B Label Structures of Similar Size but Different Shape in Super-Resolution Imaging

Subcellular structures containing autophagy-related proteins of the Atg8 protein family have been investigated with conventional wide-field fluorescence and single molecule localisation microscopy. Fusion proteins of GABARAP and LC3B, respectively, with EYFP were overexpressed in HEK293 cells. While size distributions of structures labelled by the two proteins were found to be similar, shape distributions appeared quite disparate, with EYFP-GABARAP favouring circular structures and elliptical structures being dominant for EYFP-LC3B. The latter also featured a nearly doubled fraction of U-shape structures. The experimental results point towards highly differential localisation of the two proteins, which appear to label structures representing distinct stages or even specific channels of vesicular trafficking pathways. Our data also demonstrate that the application of super-resolution techniques expands the possibilities of fluorescence-based methods in autophagy studies and in some cases can rectify conclusions obtained from conventional fluorescence microscopy with diffraction-limited resolution

<i>E</i>_GSH and pH of wild type cells grown in SC_URA to exponential phase and treated with various stressors for 1 hour.

<p><i>E</i>_GSH and pH of wild type cells grown in SC_URA to exponential phase and treated with various stressors for 1 hour.</p

Effect of heat shock on compartmental <i>E</i>_GSH and pH.

<p>A) Wild-type cells with the HSE2-lacZ (heat-shock response induction reporter) construct were pregrown in SC_URA (48 h; 30°C; 600 rpm) and then inoculated in SC_URA (A₆₀₀ = 0.001) and grown (25°C; 600 rpm) until exponential phase (A₆₀₀ = ∼0.5). Cells were then shifted to 42°C for 60 min and harvested. β-galactosidase and total protein (Bradford) assays were carried out on cell pellets and specific activity determined. B-C) Cells were pregrown in SC_URA (48 h; 30°C; 600 rpm), inoculated in SC_URA (A₆₀₀ = 0.001) and grown (25°C; 600 rpm) until exponential phase (A₆₀₀ = ∼0.5). Cells were then shifted to 42°C for 60 min and <i>E</i>_GSH B) and pH C) analyzed via flow cytometry. 10,000 cells were counted for each condition. Each experiment was conducted in triplicate with error bars representing the standard deviation of three experiments.</p

Dynamic recovery of cells after hydrogen peroxide treatment.

<p>Cells were pregrown in SC_URA (48 h; 30°C; 600 rpm), inoculated in SC_URA (A₆₀₀ = 0.001) and grown (25°C; 600 rpm) until exponential phase (A₆₀₀ = ∼0.5). Starting <i>E</i>_GSH was measured then cells were treated with hydrogen peroxide (2 mM; 20 min; 25°C), washed and resuspended in fresh medium, and pH and redox state analyzed by flow cytometry at two-minute intervals. A) cytosolic pH and <i>E</i>_GSH, B) mitochondrial matrix pH and <i>E</i>_GSH and C) peroxisomal pH and <i>E</i>_GSH. 10,000 cells were counted for each sample. Each experiment was conducted in triplicate with error bars representing the standard deviation of three experiments. The E_GSH and pH in untreated (control cells) is indicated by the marked dashed lines. Some error bars may be difficult to visualise as the errors are in the ∼5 mV range.</p

Confocal microscopy images of yeast cells expressing cytosolic, mitochondrial matrix or peroxisomal pHluorin.

<p>A) Wild type cells were pregrown in SC_URA (48 h; 30°C; 600 rpm), inoculated into SC_URA (A₆₀₀ = 0.001), grown until exponential phase and examined using an Olympus FV-1000 confocal microscope at a magnification of 100X. B) In cells where mitochondrial DNA was visualized cells were incubated with DAPI for 30 min, washed with sterile water and examined. C) <i>pex5</i> cells were pregrown in SC_URA (48 h; 30°C; 600 rpm), inoculated into SC_URA (A₆₀₀ = 0.001), grown to exponential phase (A₆₀₀ = ∼0.5) and examined using an Olympus V-1000 confocal microscope at a magnification of 100X.</p

Representative dot plot of cells expressing cytosolic roGFP2.

<p>Cells expressing cytosolic roGFP2 were grown to exponential phase (A₆₀₀∼0.5) in SC_URA and analyzed by flow cytometry. Dot plot of wild-type cells untreated and treated with oxidant (4 mM hydrogen peroxide). Note the shift in the populations after treatment with hydrogen peroxide towards 405 nm. Dot plots were generated using FlowJo™ software.</p