Regulation of Peroxisome Homeostasis by Post-Translational Modification in the Methylotrophic Yeast Komagataella phaffii

Abstract

The methylotrophic yeast Komagataella phaffii (synoym Pichia pastoris) can grow on methanol with an associated proliferation of peroxisomes, which are subsequently degraded by pexophagy upon depletion of methanol. Two cell wall integrity and stress response component (WSC) family proteins (Wsc1 and Wsc3) sense the extracellular methanol concentration and transmit the methanol signal to Rom2. This stimulates the activation of transcription factors (Mxr1, Trm1, and Mit1 etc.), leading to the induction of methanol-metabolizing enzymes (methanol-induced gene expression) and synthesis of huge peroxisomes. Methanol-induced gene expression is repressed by the addition of ethanol (ethanol repression). This repression is not conducted directly by ethanol but rather by acetyl-CoA synthesized from ethanol by sequential reactions, including alcohol and aldehyde dehydrogenases, and acetyl-CoA synthetase. During ethanol repression, Mxr1 is inactivated by phosphorylation. Peroxisomes are degraded by pexophagy on depletion of methanol and this event is triggered by phosphorylation of Atg30 located at the peroxisome membrane. In the presence of methanol, Wsc1 and Wsc3 repress pexophagy by transmitting the methanol signal via the MAPK cascade to the transcription factor Rlm1, which induces phosphatases involved in dephosphorylation of Atg30. Upon methanol consumption, repression of Atg30 phosphorylation is released, resulting in initiation of pexophagy. Physiological significance of these machineries involved in peroxisome homeostasis and their post-translational modification is also discussed in association with the lifestyle of methylotrophic yeast in the phyllosphere.

Keywords: MAPK cascade; WSC family proteins; ethanol repression; methanol-induced gene expression; pexophagy; phosphorylation.

FIGURE 1

Regulation of peroxisome homeostasis by a methanol-sensing pathway mediated by WSC family proteins in the phyllosphere. WSC family proteins (Wsc1 and Wsc3) sense the change in methanol concentration, and transmit a signal to Rom2 which is then transmitted via two unidentified molecules (possibly Rho1 and Pkc1) to the transcription factors (TFs) Trm1, Mit1, and Mxr1. The transcription factors activate methanol-induced genes and this results in peroxisomes (Ps) biogenesis (Lin-Cereghino et al., 2006; Parua et al., 2012; Sahu et al., 2014; Wang et al., 2016; Ohsawa et al., 2018). Simultaneously, the methanol signal under Wsc1 and Rom2 is transmitted to the MAPK cascade, which includes Mpk1. The transcription factor, Rlm1 then represses pexophagy via expression of two phosphatases, PTP2A and MSG5, which regulate the level of phosphorylation (P) of the Atg30 pexophagy receptor. As depletion of methanol releases the biogenesis of peroxisomes and repression of pexophagy, the phosphorylation of KpAtg30 by Hrr25 is enhanced (Zientara-Rytter et al., 2018). In the phyllosphere, plant leaf methanol concentration oscillates diurnally, being higher in the dark period and lower in the light period (∼0–0.3%) (Kawaguchi et al., 2011). In the dark period (left panel), methanol enhances induction of peroxisome biogenesis and represses pexophagy. In the light period (right panel), methanol-induced gene expression is low, and peroxisomes are degraded via pexophagy.

FIGURE 2

Repression of methanol-induced peroxisome proliferation by the co-presence of ethanol. Ethanol is metabolized to Acetyl-CoA by the sequential reactions catalyzed by Adh2, Ald4, Acs1 and Acs2. Acetyl-CoA then represses methanol-induced gene expression. As in S. cerevisiae, acetyl-CoA may be utilized for acetylation of histones or other transcription factors that repress the expression of methanol-induced genes (Takahashi et al., 2006). Mxr1 is a key transcription factor for methanol-induced gene expression. The presence of methanol, or removal of glucose, changes the localization of Mxr1 from the cytoplasm to the nucleus, where it can bind to the promoter regions of methanol-induced genes. In the presence of ethanol, an unidentified kinase inactivates Mxr1 by phosphorylation at the serine residue S215, which causes the interaction with 14-3-3 protein in the nucleus. This interaction inhibits the function of KpMxr1 activation domain (Parua et al., 2012) (left panel). This process occurs independently of the acetyl-CoA pathway component. Changing to methanol media or depleting ethanol stimulates the dephosphorylated Mxr1 to activate the expression of methanol-induced genes. To release the ethanol repression effectively, Acs1 and Acs2 are degraded by autophagy (right panel).