A novel fluorescence-activated cell sorting (FACS)-based screening identified ATG14, the gene required for pexophagy in the methylotrophic yeast.

Pexophagy is a type of autophagy that selectively degrades peroxisomes and can be classified as either macropexophagy or micropexophagy. During macropexophagy, individual peroxisomes are sequestered by pexophagosomes and transported to the vacuole for degradation, while in micropexophagy, peroxisomes are directly engulfed by the septated vacuole. To date, some autophagy-related genes (ATGs) required for pexophagy have been identified through plate-based assays performed primarily under micropexophagy-induced conditions. Here, we developed a novel high-throughput screening system using fluorescence-activated cell sorting (FACS) to identify genes required for macropexophagy. Using this system, we discovered KpATG14, a gene that could not be identified previously in the methylotrophic yeast Komagataella phaffii due to technical limitations. Microscopic and immunoblot analyses found that KpAtg14 was required for both macropexophagy and micropexophagy. We also revealed that KpAtg14 was necessary for recruitment of the downstream factor KpAtg5 at the pre-autophagosomal structure (PAS), and consequently, for bulk autophagy. We anticipate our assay to be used to identify novel genes that are exclusively required for macropexophagy, leading to better understanding of the physiological significance of the existing two types of autophagic degradation pathways for peroxisomes.

Phosphoregulation of the transcription factor Mxr1 plays a crucial role in the concentration-regulated methanol induction in Komagataella phaffii.

Methylotrophic yeasts can utilize methanol as the sole carbon and energy source, and the expression of their methanol-induced genes is regulated based on the environmental methanol concentration. Our understanding of the function of transcription factors and Wsc family of proteins in methanol-induced gene expression and methanol sensing is expanding, but the methanol signal transduction mechanism remains undetermined. Our study has revealed that the transcription factor KpMxr1 is involved in the concentration-regulated methanol induction (CRMI) in Komagataella phaffii (Pichia pastoris) and that the phosphorylation state of KpMxr1 changes based on methanol concentration. We identified the functional regions of KpMxr1 and determined its multiple phosphorylation sites. Non-phosphorylatable substitution mutations of these newly identified phosphorylated threonine and serine residues resulted in significant defects in CRMI. We revealed that KpMxr1 receives the methanol signal from Wsc family proteins via KpPkc1 independent of the mitogen-activated protein kinase (MAPK) cascade and speculate that the activity of KpPkc1 influences KpMxr1 phosphorylation state. We propose that the CRMI pathway from Wsc to KpMxr1 diverges from KpPkc1 and that phosphoregulation of KpMxr1 plays a crucial role in CRMI.

The methanol sensor Wsc1 and MAPK Mpk1 suppress degradation of methanol-induced peroxisomes in methylotrophic yeast.

In nature, methanol is produced during the hydrolysis of pectin in plant cell walls. Methanol on plant leaves shows circadian dynamics, to which methanol-utilizing phyllosphere microorganisms adapt. In the methylotrophic yeast Komagataella phaffii (Kp; also known as Pichia pastoris), the plasma membrane protein KpWsc1 senses environmental methanol concentrations and transmits this information to induce the expression of genes for methanol metabolism and the formation of huge peroxisomes. In this study, we show that KpWsc1 and its downstream MAPK, KpMpk1, negatively regulate pexophagy in the presence of methanol concentrations greater than 0.15%. Although KpMpk1 was not necessary for expression of methanol-inducible genes and peroxisome biogenesis, KpMpk1, the transcription factor KpRlm1 and phosphatases were found to suppress pexophagy by controlling phosphorylation of KpAtg30, the key factor in regulation of pexophagy. We reveal at the molecular level how the single methanol sensor KpWsc1 commits the cell to peroxisome synthesis and degradation according to the methanol concentration, and we discuss the physiological significance of regulating pexophagy for survival in the phyllosphere. This article has an associated First Person interview with Shin Ohsawa, joint first author of the paper.

Interaction between C1-microorganisms and plants: contribution to the global carbon cycle and microbial survival strategies in the phyllosphere.

C1-microorganisms that can utilize C1-compounds, such as methane and methanol, are ubiquitous in nature, and contribute to drive the global carbon cycle between two major greenhouse gases, CO2 and methane. Plants emit C1-compounds from their leaves and provide habitats for C1-microorganisms. Among C1-microorganisms, Methylobacterium spp., representative of methanol-utilizing methylotrophic bacteria, predominantly colonize the phyllosphere and are known to promote plant growth. This review summarizes the interactions between C1-mircroorganisms and plants that affect not only the fixation of C1-compounds produced by plants but also CO2 fixation by plants. We also describe our recent understanding of the survival strategy of C1-microorganisms in the phyllosphere and the application of Methylobacterium spp. to improve rice crop yield.

A peroxisome deficiency-induced reductive cytosol state up-regulates the brain-derived neurotrophic factor pathway.

The peroxisome is a subcellular organelle that functions in essential metabolic pathways, including biosynthesis of plasmalogens, fatty acid ß-oxidation of very-long-chain fatty acids, and degradation of hydrogen peroxide. Peroxisome biogenesis disorders (PBDs) manifest as severe dysfunction in multiple organs, including the central nervous system (CNS), but the pathogenic mechanisms in PBDs are largely unknown. Because CNS integrity is coordinately established and maintained by neural cell interactions, we here investigated whether cell-cell communication is impaired and responsible for the neurological defects associated with PBDs. Results from a noncontact co-culture system consisting of primary hippocampal neurons with glial cells revealed that a peroxisome-deficient astrocytic cell line secretes increased levels of brain-derived neurotrophic factor (BDNF), resulting in axonal branching of the neurons. Of note, the BDNF expression in astrocytes was not affected by defects in plasmalogen biosynthesis and peroxisomal fatty acid ß-oxidation in the astrocytes. Instead, we found that cytosolic reductive states caused by a mislocalized catalase in the peroxisome-deficient cells induce the elevation in BDNF secretion. Our results suggest that peroxisome deficiency dysregulates neuronal axogenesis by causing a cytosolic reductive state in astrocytes. We conclude that astrocytic peroxisomes regulate BDNF expression and thereby support neuronal integrity and function.

Fatty acid composition of the methylotrophic yeast Komagataella phaffii grown under low- and high-methanol conditions.

In this study, we analysed the intracellular fatty acid profiles of Komagataella phaffii during methylotrophic growth. K. phaffii grown on methanol had significantly lower total fatty acid contents in the cells compared with glucose-grown cells. C18 and C16 fatty acids were the predominant fatty acids in K. phaffii, although the contents of odd-chain fatty acids such as C17 fatty acids were also relatively high. Moreover, the intracellular fatty acid composition of K. phaffii changed in response to not only carbon sources but also methanol concentrations: C17 fatty acids and C18:2 content increased significantly as methanol concentration increased, whereas C18:1 and C18:3 contents were significantly lower in methanol-grown cells. The intracellular content of unidentified compounds (Cn H2n O4 ), on the other hand, was significantly greater in cells grown on methanol. As the intracellular contents of these Cn H2n O4 compounds were significantly higher in a gene-disrupted strain for glutathione peroxidase (gpx1Δ) than in the wild-type strain, we presume that the Cn H2n O4 compounds are fatty acid peroxides. These results indicate that K. phaffii can coordinate intracellular fatty acid composition during methylotrophic growth in order to adapt to high-methanol conditions and that certain fatty acid species such as C17:0, C17:1, C17:2 and C18:2 may be related to the physiological functions by which K. phaffii adapts to high-methanol conditions.

Yeast Hog1 proteins are sequestered in stress granules during high-temperature stress.

The yeast high-osmolarity glycerol (HOG) pathway plays a central role in stress responses. It is activated by various stresses, including hyperosmotic stress, oxidative stress, high-temperature stress and exposure to arsenite. Hog1, the crucial MAP kinase of the pathway, localizes to the nucleus in response to high osmotic concentrations, i.e. high osmolarity; but, otherwise, little is known about its intracellular dynamics and regulation. By using the methylotrophic yeast Candida boidinii, we found that CbHog1-Venus formed intracellular dot structures after high-temperature stress in a reversible manner. Microscopic observation revealed that CbHog1-mCherry colocalized with CbPab1-Venus, a marker protein of stress granules. Hog1 homologs in Pichia pastoris and Schizosaccharomyces pombe also exhibited similar dot formation under high-temperature stress, whereas Saccharomyces cerevisiae Hog1 (ScHog1)-GFP did not. Analysis of CbHog1-Venus in C. boidinii revealed that a ß-sheet structure in the N-terminal region was necessary and sufficient for its localization to stress granules. Physiological studies revealed that sequestration of activated Hog1 proteins in stress granules was responsible for downregulation of Hog1 activity under high-temperature stress.This article has an associated First Person interview with the first author of the paper.

Three Distinct Types of Microautophagy Based on Membrane Dynamics and Molecular Machineries.

Microautophagy is originally defined as lysosomal (vacuolar) membrane dynamics to directly enwrap and transport cytosolic components into the lumen of the lytic organelle. Molecular details of microautophagy had remained unknown until genetic studies in yeast identified a set of proteins required for the process. Subsequent studies with other experimental model organisms resulted in a series of discoveries that accompanied an expansion of the definition of microautophagy to also encompass endosomal membrane dynamics. These findings, however, still impose puzzling, non-integrated images as to the molecular mechanism of microautophagy. By reviewing recent studies on microautophagy in various experimental systems, we propose the classification of microautophagy into three types, as the basis for developing a comprehensive view of the process.

Methanol production by reversed methylotrophy constructed in Escherichia coli.

We constructed a reversed methylotrophic pathway that produces methanol, a promising feedstock for production of useful compounds, from fructose 6-phosphate (F6P), which can be supplied by catabolism of biomass-derived sugars including glucose, by a synthetic biology approach. Using Escherichia coli as an expression host, we heterologously expressed genes encoding methanol utilization enzymes from methylotrophic bacteria, i.e. the NAD+-dependent methanol dehydrogenase (MDH) from Bacillus methanolicus S1 and an artificial fusion enzyme of 3-hexulose-6-phosphate synthase and 6-phospho-3-hexuloisomerase from Mycobacterium gastri MB19 (HPS-PHI). We confirmed that these enzymes can catalyze reverse reactions of methanol oxidation and formaldehyde fixation. The engineered E. coli strain co-expressing MDH and HPS-PHI genes produced methanol in resting cell reactions not only from F6P but also from glucose. We successfully conferred reversed methylotrophy to E. coli and our results provide a proof-of-concept for biological methanol production from biomass-derived sugar compounds.

Methylotrophic Yeasts: Current Understanding of Their C1-Metabolism and its Regulation by Sensing Methanol for Survival on Plant Leaves.

Methylotrophic yeasts, which are able to utilize methanol as the sole carbon and energy source, have been intensively studied in terms of physiological function and practical applications. When these yeasts grow on methanol, the genes encoding enzymes and proteins involved in methanol metabolism are strongly induced. Simultaneously, peroxisomes, organelles that contain the key enzymes for methanol metabolism, massively proliferate. These characteristics have made methylotrophic yeasts efficient hosts for heterologous protein production using strong and methanol-inducible gene promoters and also model organisms for the study of peroxisome dynamics. Much attention has been paid to the interaction between methylotrophic microorganisms and plants. In this chapter, we describe how methylotrophic yeasts proliferate and survive on plant leaves, focusing on their physiological functions and lifestyle in the phyllosphere. Our current understanding of the molecular basis of methanol-inducible gene expression, including methanol-sensing and its applications, is also summarized.

Engineering the expression system for Komagataella phaffii (Pichia pastoris): an attempt to develop a methanol-free expression system.

The construction of a methanol-free expression system of Komagataella phaffii (Pichia pastoris) was attempted by engineering a strong methanol-inducible DAS1 promoter using Citrobacter braakii phytase production as a model case. Constitutive expression of KpTRM1, formerly PRM1-a positive transcription regulator for methanol-utilization (MUT) genes of K. phaffii,was demonstrated to produce phytase without addition of methanol, especially when a DAS1 promoter was used but not an AOX1 promoter. Another positive regulator, Mxr1p, did not have the same effect on the DAS1 promoter, while it was more effective than KpTrmp1 on the AOX1 promoter. Removing a potential upstream repression sequence (URS) and multiplying UAS1DAS1 in the DAS1 promoter significantly enhanced the yield of C. braakii phytase with methanol-feeding, which surpassed the native AOX1 promoter by 80%. However, multiplying UAS1DAS1 did not affect the yield of methanol-free expression by constitutive KpTrm1p. Another important region to enhance the effect of KpTrm1p under a methanol-free condition was identified in the DAS1 promoter, and was termed ESPDAS1. Nevertheless, methanol-free phytase production using an engineered DAS1 promoter outperformed phytase production with the GAP promoter by 25%. Difference in regulation by known transcription factors on the AOX1 promoter and the DAS1 promoter was also illustrated.

Pantothenate auxotrophy of Methylobacterium spp. isolated from living plants.

A number of pink-pigmented facultative methylotrophs (PPFMs) belonging to Methylobacterium spp. isolated from living plant samples were found to require B vitamins for their growth in minimal medium, and most B vitamin-auxotrophic PPFMs required pantothenate (vitamin B5). Further investigation of pantothenate auxotrophy using the representative strain Methylobacterium sp. OR01 demonstrated that this strain cannot synthesize ß-alanine, one of the precursors of pantothenate. ß-alanine and several precursors of pantothenate restored the growth of Methylobacterium sp. OR01 in minimal medium. Furthermore, this strain could colonize leaves of Arabidopsis thaliana cultivated in medium without pantothenate or its precursors. Pantothenate, ß-alanine and several precursors were detected in the suspension of A. thaliana leaves. These results suggest that pantothenate-auxotrophic PPFMs can symbiotically colonize the surface of plant leaves by acquiring ß-alanine and other precursors, in addition to pantothenate. Finally, the fitness advantage of B vitamin auxotrophy of PPFMs in the phyllosphere environment is discussed.

Novel function of Wsc proteins as a methanol-sensing machinery in the yeast Pichia pastoris.

Wsc family proteins are plasma membrane spanning sensor proteins conserved from yeasts to mammalian cells. We studied the functional roles of Wsc family proteins in the methylotrophic yeast Pichia pastoris, and found that PpWsc1 and PpWsc3 function as methanol-sensors during growth on methanol. PpWsc1 responds to a lower range of methanol concentrations than PpWsc3. PpWsc1, but not PpWsc3, also functions during high temperature stress, but PpWsc1 senses methanol as a signal that is distinct from high-temperature stress. We also found that PpRom2, which is known to function downstream of the Wsc family proteins in the cell wall integrity pathway, was also involved in sensing methanol. Based on these results, these PpWsc family proteins were demonstrated to be involved in sensing methanol and transmitting the signal via their cytoplasmic tail to the nucleus via PpRom2, which plays a critical role in regulating expression of a subset of methanol-inducible genes to coordinate well-balanced methanol metabolism.

A Pichia pastoris single-cell biosensor for detection of enzymatically produced methanol.

We conducted single-cell analyses of the methylotrophic yeast Pichia pastoris to develop a biosensor for the detection of methanol produced by heterologous enzymes. In this biosensor, methanol and its subsequent metabolism induce expression of a gene encoding a fluorescent protein that was placed under the control of a methanol-inducible promoter. Using quantitative analyses of fluorescence microscopy images, a methanol-inducible promoter and a host strain were selected, and preculture and assay conditions were optimized to improve the methanol detection limit. Fluorescence-activated cell sorting (FACS) analysis of the distribution and geometric mean of cellular fluorescence intensity against various concentrations of methanol revealed a detection limit of 2.5 µM. Finally, this biosensor was applied to evaluate the activity of a heterologously expressed pectin methylesterase (PME). The cellular fluorescence intensity was proportional to the copy number of the PME expression cassette, the protein level, and the enzyme activity. This biosensor can be used for high-throughput screening of single cells harboring high methanol-producing activity, and thereby, the development of a bioconversion process using methanol-producing enzymes.

Role of Acyl Chain Composition of Phosphatidylcholine in Tafazzin-Mediated Remodeling of Cardiolipin in Liposomes.

Remodeling of the acyl chain compositions of cardiolipin (CL) species by the transacylase tafazzin is an important process for maintaining optimal mitochondrial functions. The results of mechanistic studies on the tafazzin-mediated transacylation from phosphatidylcholine (PC) to monolyso-CL (MLCL) in artificial lipid membranes are controversial. The present study investigated the role of the acyl chain composition of PC in the Saccharomyces cerevisiae tafazzin-mediated remodeling of CL by examining the structural factors responsible for the superior acyl donor ability of dipalmitoleoyl (16:1) PC over dipalmitoyl (16:0) PC. To this end, we synthesized systematic derivatives of dipalmitoleoyl PC; for example, the location of the cis double bond was migrated from the Δ9-position toward either end of the acyl chains (the Δ5- or Δ13-position), the cis double bond in the sn-1 or sn-2 position or both, was changed to a trans form, and palmitoleoyl and palmitoyl groups were exchanged in the sn-1 and sn-2 positions, maintaining similar PC fluidities. Analyses of the tafazzin-mediated transacylation from these PCs to sn-2'-MLCL(18:1-18:1/18:1-OH) in the liposomal membrane revealed that tafazzin strictly discriminates the molecular configuration of the acyl chains of PCs, including their glycerol positions (sn-1 or sn-2); however, the effects of PC fluidity on the reaction may not be neglected. On the basis of the findings described herein, we discuss the relevance of the so-called thermodynamic remodeling hypothesis that presumes no acyl selectivity of tafazzin.

Synthesized Aß42 Caused Intracellular Oxidative Damage, Leading to Cell Death, via Lysosome Rupture.

Neuronal cellular accumulation of amyloid beta peptide (Aß) has been implicated in the pathogenesis of Alzheimer's disease (AD). Intracellular accumulation of Aß42, a toxic form of Aß, was observed as an early event in AD patients. However, its contribution and the cellular mechanism of cell death remained unclear. We herein revealed the mechanism by which Aß42 incorporated into cells leads to cell death by using chemically synthesized Aß42 variants. The Aß42 variant Aß42 (E22P) which has an increased tendency to oligomerize, accumulated in lysosomes at an earlier stage than wild-type Aß42, leading to higher ROS production and lysosomal membrane oxidation, and resulting in cell death. On the other hand, Aß42 (E22V), which is incapable of oligomerization, did not accumulate in cells or affect the cell viability. Moreover, intracellular localization of EGFP-Galectin-3, a ß-galactoside binding lectin, showed that accumulation of oligomerized Aß42 in lysosomes caused lysosomal membrane permeabilization (LMP). Overexpression of lysosome-localized LAMP1-fused peroxiredoxin 1 and treatment with U18866A, an inhibitor of cholesterol export from lysosomes that causes an increase in lysosomal membrane stability, attenuated Aß42-mediated LMP and cell death. Our findings show that lysosomal ROS generation by toxic conformer of Aß led to cell death via LMP, and suggest that these events are potential targets for AD prevention.Key words: Amyloid-beta (Aß), Cell death, Lysosome, Lysosomal membrane permeabilization, Reactive oxygen species (ROS).

Mechanism for Remodeling of the Acyl Chain Composition of Cardiolipin Catalyzed by Saccharomyces cerevisiae Tafazzin.

Remodeling of the acyl chains of cardiolipin (CL) is responsible for final molecular composition of mature CL after de novo CL synthesis in mitochondria. Yeast Saccharomyces cerevisiae undergoes tafazzin-mediated CL remodeling, in which tafazzin serves as a transacylase from phospholipids to monolyso-CL (MLCL). In light of the diversity of the acyl compositions of mature CL between different organisms, the mechanism underlying tafazzin-mediated transacylation remains to be elucidated. We investigated the mechanism responsible for transacylation using purified S. cerevisiae tafazzin with liposomes composed of various sets of acyl donors and acceptors. The results revealed that tafazzin efficiently catalyzes transacylation in liposomal membranes with highly ordered lipid bilayer structure. Tafazzin elicited unique acyl chain specificity against phosphatidylcholine (PC) as follows: linoleoyl (18:2) > oleoyl (18:1) = palmitoleoyl (16:1) ≫ palmitoyl (16:0). In these reactions, tafazzin selectively removed the sn-2 acyl chain of PC and transferred it into the sn-1 and sn-2 positions of MLCL isomers at equivalent rates. We demonstrated for the first time that MLCL and dilyso-CL have inherent abilities to function as an acyl donor to monolyso-PC and acyl acceptor from PC, respectively. Furthermore, a Barth syndrome-associated tafazzin mutant (H77Q) was shown to completely lack the catalytic activity in our assay. It is difficult to reconcile the present results with the so-called thermodynamic remodeling hypothesis, which premises that tafazzin reacylates MLCL by unsaturated acyl chains only in disordered non-bilayer lipid domain. The acyl specificity of tafazzin may be one of the factors that determine the acyl composition of mature CL in S. cerevisiae mitochondria.

Pexophagy in yeasts.

Pexophagy, selective degradation of peroxisomes via autophagy, is the main system for reducing organelle abundance. Elucidation of the molecular machinery of pexophagy has been pioneered in studies of the budding yeast Saccharomyces cerevisiae and the methylotrophic yeasts Pichia pastoris and Hansenula polymorpha. Recent analyses using these yeasts have elucidated the molecular machineries of pexophagy, especially in terms of the interactions and modifications of the so-called adaptor proteins required for guiding autophagic membrane biogenesis on the organelle surface. Based on the recent findings, functional relevance of pexophagy and another autophagic pathway, mitophagy (selective autophagy of mitochondria), is discussed. We also discuss the physiological importance of pexophagy in these yeast systems.

Tor and the Sin3-Rpd3 complex regulate expression of the mitophagy receptor protein Atg32 in yeast.

When mitophagy is induced in Saccharomyces cerevisiae, the mitochondrial outer membrane protein ScAtg32 interacts with the cytosolic adaptor protein ScAtg11. ScAtg11 then delivers the mitochondria to the pre-autophagosomal structure for autophagic degradation. Despite the importance of ScAtg32 for mitophagy, the expression and functional regulation of ScAtg32 are poorly understood. In this study, we identified and characterized the ScAtg32 homolog in Pichia pastoris (PpAtg32). Interestingly, we found that PpAtg32 was barely expressed before induction of mitophagy and was rapidly expressed after induction of mitophagy by starvation. Additionally, PpAtg32 was phosphorylated when mitophagy was induced. We found that PpAtg32 expression was suppressed by Tor and the downstream PpSin3-PpRpd3 complex. Inhibition of Tor by rapamycin induced PpAtg32 expression, but could neither phosphorylate PpAtg32 nor induce mitophagy. Based on these findings, we conclude that the Tor and PpSin3-PpRpd3 pathway regulates PpAtg32 expression, but not PpAtg32 phosphorylation.

Unique C-terminal region of Hap3 is required for methanol-regulated gene expression in the methylotrophic yeast Candida boidinii.

The Hap complex of the methylotrophic yeast Candida boidinii was found to be required for methanol-regulated gene expression. In this study, we performed functional characterization of CbHap3p, one of the Hap complex components in C. boidinii. Sequence alignment of Hap3 proteins revealed the presence of a unique extended C-terminal region, which is not present in Hap3p from Saccharomyces cerevisiae (ScHap3p), but is found in Hap3p proteins of methylotrophic yeasts. Deletion of the C-terminal region of CbHap3p (Δ256-292 or Δ107-237) diminished activation of methanol-regulated genes and abolished the ability to grow on methanol, but did not affect nuclear localization or DNA-binding ability. However, deletion of the N-terminal region of CbHap3p (Δ1-20) led to not only a growth defect on methanol and a decreased level of methanol-regulated gene expression, but also impaired nuclear localization and binding to methanol-regulated gene promoters. We also revealed that CbHap3p could complement the growth defect of the Schap3Δ strain on glycerol, although ScHap3p could not complement the growth defect of a Cbhap3Δ strain on methanol. We conclude that the unique C-terminal region of CbHap3p contributes to maximum activation of methanol-regulated genes, whilst the N-terminal region is required for nuclear localization and binding to DNA.