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.

In methylotrophic yeasts, the expression of methanol-inducible genes is repressed by ethanol even in the presence of methanol, a phenomenon called ethanol repression. The mechanism of ethanol repression in Komagataella phaffii (Pichia pastoris) was studied, and acetyl-CoA synthesis from ethanol by sequential reactions of alcohol dehydrogenase, aldehyde dehydrogenase and acetyl-CoA synthetase (ACS) was involved in ethanol repression. Molecular analysis of the ACS-encoding gene product KpAcs1 revealed that its N-terminal motif, which is conserved in methylotrophic yeasts, was required for ethanol repression. ACS activity was downregulated during methanol-induced gene expression, which partially depended on autophagy. In addition, acetyl-CoA synthesis and phosphorylation of a transcription factor KpMxr1 were found to contribute to ethanol repression in a synergistic manner.

In nature, methanol is produced during the hydrolysis of pectin in plant cell walls. Methanol shows circadian dynamics on plant leaves to which methanol-utilizing phyllosphere microorganisms adapt. In the methylotrophic yeast Komagataella phaffii (Pichia pastoris), the plasma membrane protein KpWsc1 senses environmental methanol concentrations, and transmits the information to induce genes for methanol metabolism together with huge peroxisomes. In this study, we show that KpWsc1 and its downstream MAPK negatively regulate pexophagy in the presence of >0.15% methanol. Although KpMpk1 was not necessary for expression of methanol-inducible genes and peroxisome biogenesis, KpMpk1, KpRlm1 and a phosphatase were found suppress pexophagy by controlling phosphorylation level of KpAtg30, the key factor 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 discuss the physiological significance of regulating pexophagy for survival in the phyllosphere.

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.

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.