Methylotrophic yeasts are able to grow on methanol and utilize it as their sole source of carbon and energy. This growth involves the proliferation of peroxisomes that contain the enzymes involved in methanol metabolism, such as alcohol oxidase (AOX), dihydroxyacetone synthase (DAS), as well as the cytosolic methanol-metabolizing enzymes, formaldehyde dehydrogenase (FLD), and formate dehydrogenase (FDH) (Sakai et al., 1996; Yurimoto et al., 2005). Methanol also strongly induces the transcript levels of methanol-metabolizing enzymes. These attributes make methylotrophic yeasts attractive model organisms in the study of the molecular mechanisms involved in peroxisome dynamics and as hosts for heterologous gene expression (Oku and Sakai, 2010).

Peroxisome dynamics in methylotrophic yeasts are tightly regulated by the available carbon source and this is reflected in the extent to which peroxisomal proteins are synthesized: the maximum-level of expression is achieved in the presence of methanol (methanol induction), a low-level of expression is observed in the absence of carbon source (derepression), and the synthesis is completely repressed by the presence of glucose (glucose repression) and ethanol (ethanol repression). In addition, cells regulate peroxisome biogenesis in response to changes in methanol concentration by altering the expression of methanol-induced genes (Kawaguchi et al., 2011; Ohsawa et al., 2017; Takeya et al., 2018). Methanol-induced peroxisomes are degraded by selective autophagy, i.e., pexophagy in response to methanol depletion, and strongly to the carbon source shift to glucose and ethanol (Tuttle and Dunn, 1995; van Zutphen et al., 2008; Nazarko et al., 2009; Oku and Sakai, 2016). Although several transcription factors that control methanol induction, derepression, and glucose and ethanol repression, have been identified and characterized (Leao-Helder et al., 2003; Lin-Cereghino et al., 2006; Sasano et al., 2008; Sasano et al., 2010; Sahu et al., 2014; Oda et al., 2015; Wang et al., 2016), their methanol-sensing mechanism and signaling pathways have yet to be fully elucidated.

Our recent studies on the methylotrophic yeast Komagataella phaffii (synonym. Pichia pastoris) revealed that cell-surface WSC family proteins act as methanol-sensing machineries and control the biogenesis and degradation of peroxisomes. In this review, we summarize the processes involved in the regulation of peroxisome homeostasis in the methylotrophic yeasts, including post-translational modifications such as protein phosphorylation, downstream signal cascades via WSC family proteins, and the molecular mechanism of ethanol repression. In addition, we discuss the physiological significance of peroxisome homeostasis for methylotrophic yeasts living in the phyllosphere environment (aerial parts of plants).

Our previous studies revealed that WSC family proteins act as methanol-sensing machinery for regulating methanol-induced gene expression in K. phaffii (Ohsawa et al., 2017). WSC family proteins were identified as “nanospring” mechanosensors of cell wall damage caused by high temperatures and hypo-osmotic stress in Saccharomyces cerevisiae (ScWsc1/ScSlg1) (Lodder et al., 1999; Dupres et al., 2009). In the K. phaffii genome, three WSC family protein-encoding genes have been identified (KpWSC1, KpWSC2, and KpWSC3). The significant features of Wsc family proteins, a cysteine-rich domain (CRD), a serine/threonine rich region (STR) in extracellular region, and a Rom2-interacting site at cytoplasmic tail together with a transmembrane domain (TMD) are conserved in all three KpWsc family proteins. In the Kpwsc1∆wsc3∆ double deletion mutant, dose-dependent (0.001–1.0%) methanol-induced gene expression (AOX, DAS, FLD, FDH) was abolished, indicating that these WSC family proteins play a role in methanol sensing (Ohsawa et al., 2017). Characterization of KpWsc1 and KpWsc3 has revealed that KpWsc1 responds to a lower range of methanol concentration (0.01–0.05%) whilst KpWsc3 responds to a higher range (0.1–0.5%) and that KpWsc1 also acts as a sensor of cell wall damage caused by high temperature stress. These properties might enable cells to respond to a wide range of methanol concentration and to two different stimuli (methanol and high temperature stress) simultaneously. As to KpWsc2, it was not related to the regulation of methanol-inducible gene expression and high temperature stress (Ohsawa et al., 2017).

Selective autophagy of organelles requires receptor proteins on the surface of the target organelle that recruit the autophagy-related (Atg) proteins to form autophagosomes (Xie and Klionsky, 2007). The receptor for pexophagy in K. phaffii is KpAtg30 (Farré et al., 2013 et al.). It interacts with the peroxins which are involved in peroxisome biogenesis (KpPex3 and KpPex14) and with three Atg proteins (KpAtg8, KpAtg11, and KpAtg37) (Farré et al., 2008; Farré et al., 2013; Nazarko et al., 2014; Burnett et al., 2015). Two phosphorylation events of KpAtg30 recruit Atg proteins. Phosphorylation sites of KpAtg30 are at serine residue S71 and S112 by an unknown kinase and KpHrr25, a homologue of casein kinase 1δ, respectively (Farré et al., 2013). The peroxisomal acyl-CoA-binding protein, KpAtg37, is recruited to the KpAtg30 in Pex3-dependnt manner. The recruitment of KpAtg37 to KpAtg30 enables binding of KpHrr25 to KpAtg30, leading the phosphorylation of KpAtg30 at S112 to recruit KpAtg11 to KpAtg30 (Zientara-Rytter et al., 2018). KpPex3 interacts with KpAtg30 at KpHrr25-binding site,_it makes a state that is unfavorable for KpHrr25 binding, leading a second phosphorylation at S71 to recruit KpAtg8 to KpAtg30. Our recent study provides further evidence of importance of phosphorylation in pexophagy; we demonstrated that KpWsc1 and its downstream MAPK negatively regulate pexophagy in the presence of methanol via repression of KpAtg30 phosphorylation (Ohsawa et al., 2021). Furthermore, KpRlm1-dependent genes encoding phosphatases (KpMsg5 and KpPtp2A) have a role in maintaining a low level of KpAtg30 phosphorylation in the presence of methanol (Ohsawa et al., 2021). In the S. cerevisiae CWI pathway, ScMpk1 activates ScRlm1 for induction of glucan synthase during cell surface stress (Rodicio and Heinisch, 2010; Levin, 2011). By analogy, we assume that KpWsc1 activates KpMpk1, and transmits a signal to KpRlm1 for suppression of pexophagy. As described above, the signaling pathway that controls peroxisome synthesis via the regulation of methanol-induced gene expression does not employ components of KpMpk1 and KpRlm1. These results suggest that the KpWsc1 methanol-sensing machinery regulates both the level of peroxisome biogenesis and the degree of pexophagy in response to the methanol concentration, by two distinct signaling pathways, MAPK-independent and MAPK-dependent pathways, respectively (Figure 1).

In the phyllosphere, the methanol concentration on plant leaves oscillates diurnally, being higher in the dark period and lower in the light period (∼0–0.3%), (Kawaguchi et al., 2011). The methylotrophic yeasts C. boidinii and K. phaffii can proliferate on plant leaves at a cell-division rate of 3-4 per 7–10 days (Kawaguchi et al., 2011). During proliferation on plant leaves, C. boidinii cells repeat the daily cycle of peroxisome biogenesis and degradation by pexophagy. During this diurnal cycle, factors for peroxisome biogenesis, methanol-utilizing enzymes, and pexophagy (e.g. CbAtg30) are essential for methylotrophic yeast cells growth. Evidently, they regulate peroxisome dynamics in response to the methanol concentration of the phyllosphere. We speculate that the methanol-sensing machinery, KpWsc1 and KpWsc3, are responsible for regulation of peroxisome homeostasis on plant leaves (Figure 1). Remarkably, non-selective (bulk) autophagy, which is dependent on Atg1 but not Atg30, occurs throughout the daily dark-light cycle (Kawaguchi et al., 2011). Conversely, Atg30-dependent pexophagy is only observed in the light period. The negative regulation of pexophagy via methanol-sensing machinery (Wsc1 and MAPK) could explain how pexophagy is selectively inhibited during the dark period (when the methanol concentration is higher), while the non-selective autophagy machinery is active throughout the daily light-dark cycle. Revealing the molecular mechanism of post-translational regulation of methanol-induced gene expression and peroxisome dynamics would facilitate the improvement of the system for heterologous gene expression and would also further our understanding of the physiological roles of these regulatory machineries as survival strategies in the phyllosphere.

Peroxisomes are highly dynamic organelles whose homeostasis is controlled by biogenesis and pexophagy in response to the extracellular environment. In the methylotrophic yeast K. phaffii, huge peroxisomes are induced during growth on methanol and subsequently degraded by pexophagy in response to both the concentration of methanol or to a change in the carbon source (glucose and ethanol). Although the molecular mechanism of peroxisome dynamics has been studied in detail, less is known about the upstream signaling pathway and those factors responsible for regulating these peroxisome dynamics at a physiological level. The methanol-signaling pathway mediated by methanol-sensing machinery of WSC family proteins has been shown to regulate peroxisome dynamics depending on the environmental methanol concentration through methanol-induced gene expression and the phospho-regulation of a pexophagy receptor, Atg30. Based on these results, we propose that the physiological significance of peroxisome homeostasis is linked to the diurnal oscillation in methanol concentration on plant leaves. We also show new molecular machinery responsible for ethanol repression of the peroxisome induction, by acetyl-CoA. Further studies on the signaling mechanisms involved in peroxisome homeostasis will facilitate a better understanding of the physiological role of the regulation of peroxisome dynamics.