Saccharomyces cerevisiae target of rapamycin (TOR) complex 2 (TORC2) is an essential regulator of plasma membrane lipid and protein homeostasis. How TORC2 activity is modulated in response to changes in the status of the cell envelope is unclear. Here we document that TORC2 subunit Avo2 is a direct target of Slt2, the mitogenactivated protein kinase (MAPK) of the cell wall integrity pathway. Activation of Slt2 by overexpression of a constitutively active allele of an upstream Slt2 activator (Pkc1) or by auxin-induced degradation of a negative Slt2 regulator (Sln1) caused hyperphosphorylation of Avo2 at its MAPK phosphoacceptor sites in a Slt2-dependent manner and diminished TORC2-mediated phosphorylation of its major downstream effector, protein kinase Ypk1. Deletion of Avo2 or expression of a phosphomimetic Avo2 allele rendered cells sensitive to two stresses (myriocin treatment and elevated exogenous acetic acid) that the cell requires Ypk1 activation by TORC2 to survive. Thus, Avo2 is necessary for optimal TORC2 activity, and Slt2-mediated phosphorylation of Avo2 down-regulates TORC2 signaling. Compared with wild-type Avo2, phosphomimetic Avo2 shows significant displacement from the plasma membrane, suggesting that Slt2 inhibits TORC2 by promoting Avo2 dissociation. Our findings are the first demonstration that TORC2 function is regulated by MAPK-mediated phosphorylation.

Komagataella phaffii (Pichia pastoris) is one of the most extensively applied yeast species in pharmaceutical and biotechnological industries, and, therefore, also called the biotech yeast. However, thanks to more advanced strain engineering techniques, it recently started to gain attention as model organism in fundamental research. So far, the most studied model yeast is its distant cousin, Saccharomyces cerevisiae. While these data are of great importance, they limit our knowledge to one organism only. Since the divergence of the two species 250 million years ago, K. phaffii appears to have evolved less rapidly than S. cerevisiae, which is why it remains more characteristic of the common ancient yeast ancestors and shares more features with metazoan cells. This makes K. phaffii a valuable model organism for research on eukaryotic molecular cell biology, a potential we are only beginning to fully exploit. As methylotrophic yeast, K. phaffii has the intriguing property of being able to efficiently assimilate methanol as a sole source of carbon and energy. Therefore, major efforts have been made using K. phaffii as model organism to study methanol assimilation, peroxisome biogenesis and pexophagy. Other research topics covered in this review range from yeast genetics including mating and sporulation behavior to other cellular processes such as protein secretion, lipid biosynthesis and cell wall biogenesis. In this review article, we compare data obtained from K. phaffii with S. cerevisiae and other yeasts whenever relevant, elucidate major differences, and, most importantly, highlight the big potential of using K. phaffii in fundamental research.

All intact cells, and their organelles, are surrounded by a ∼30 Å hydrophobic film that typically separates the interior from the environment. This film is composed of lipid bilayers that form from a pool of structurally highly diverse, amphipathic lipids. The specific composition and nature of these lipids strongly contributes to many different processes in the cell by influencing membrane structures, membrane protein sorting and functionalities. In this review, we discuss strategies to alter membrane lipid compositions of organelles and plasma membranes in different organisms, focusing on microbial cells. Reflecting the many essential roles of lipids in cellular regulation, we delineate diverse cellular processes affected by membrane lipid modifications and discuss possible applications in a biotechnological and biomedical context. A major motivation for membrane lipid engineering has been the improvement of expression, translocation and activity of heterologous membrane proteins, which can facilitate the biochemical and structural characterization of this challenging class of proteins. Additionally, better expression of membrane proteins or membrane lipid engineering - or a combination of both - led to improved production of high-value compounds and food additives, e.g. polyunsaturated fatty acids and glycolipids, in diverse hosts. More recently it has been shown that diverse cellular pathologies such as cancer and Alzheimer's disease are associated with lipid alterations. Hence, the progress in our understanding of membrane structure, function and protein-lipid interactions, and the resulting possibilities regarding the engineering of membrane lipid composition clearly enable novel nutraceutical and pharmaceutical interventions to be developed. Significant progress in this important area of research is highlighted in this review.

Eukaryotic cell survival requires maintenance of plasma membrane (PM) homeostasis in response to environmental insults and changes in lipid metabolism. In yeast, a key regulator of PM homeostasis is target of rapamycin (TOR) complex 2 (TORC2), a multiprotein complex containing the evolutionarily conserved TOR protein kinase isoform Tor2. PM localization is essential for TORC2 function. One core TORC2 subunit (Avo1) and two TORC2-­associated regulators (Slm1 and Slm2) contain pleckstrin homology (PH) domains that exhibit specificity for binding phosphatidylinositol-4,5- bisphosphate (PtdIns4,5P2). To investigate the roles of PtdIns4,5P2 and constituent subunits of TORC2, we used auxin-inducible degradation to systematically eliminate these factors and then examined localization, association, and function of the remaining TORC2 components. We found that PtdIns4,5P2 depletion significantly reduced TORC2 activity, yet did not prevent PM localization or cause disassembly of TORC2. Moreover, truncated Avo1 (lacking its C-terminal PH domain) was still recruited to the PM and supported growth. Even when all three PH-containing proteins were absent, the remaining TORC2 subunits were PM-bound. Revealingly, Avo3 localized to the PM independent of both Avo1 and Tor2, whereas both Tor2 and Avo1 required Avo3 for their PM anchoring. Our findings provide new mechanistic information about TORC2 and pinpoint Avo3 as pivotal for TORC2 PM localization and assembly in vivo.

To observe internalization of the yeast pheromone receptor Ste2 by fluorescence microscopy in live cells in real time, we visualized only those molecules present at the cell surface at the time of agonist engagement (rather than the total cellular pool) by tagging this receptor at its N-terminus with an exocellular fluorogen-activating protein (FAP). A FAP is a single-chain antibody engineered to bind tightly a nonfluorescent, cell-impermeable dye (fluorogen), thereby generating a fluorescent complex. The utility of FAP tagging to study trafficking of integral membrane proteins in yeast, which possesses a cell wall, had not been examined previously. A diverse set of signal peptides and propeptide sequences were explored to maximize expression. Maintenance of the optimal FAP-Ste2 chimera intact required deletion of two, paralogous, glycosylphosphatidylinositol (GPI)-anchored extracellular aspartyl proteases (Yps1 and Mkc7). FAP-Ste2 exhibited a much brighter and distinct plasma membrane signal than Ste2-GFP or Ste2-mCherry yet behaved quite similarly. Using FAP-Ste2, new information was obtained about the mechanism of its internalization, including novel insights about the roles of the cargo-selective endocytic adaptors Ldb19/Art1, Rod1/Art4, and Rog3/Art7.

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Targeted gene knockouts play an important role in the study of gene function. For the generation of knockouts in the industrially important yeast Pichia pastoris, several protocols have been published to date. Nevertheless, creating a targeted knockout in P. pastoris still is a time‐consuming process, as the existing protocols are labour intensive and/or prone to accumulate nucleotide mutations. In this study, we introduce a novel, user‐friendly vector‐based system for the generation of targeted knockouts in P. pastoris. Upon confirming the successful knockout, respective selection markers can easily be recycled. Excision of the marker is mediated by Flippase (Flp) recombinase and occurs at high frequency (≥95%). We validated our knockout system by deleting 20 (confirmed and putative) protease genes and five genes involved in biosynthetic pathways. For the first time, we describe gene deletions of PRO3 and PHA2 in P. pastoris, genes involved in proline, and phenylalanine biosynthesis, respectively. Unexpectedly, knockout strains of PHA2 did not display the anticipated auxotrophy for phenylalanine but rather showed a bradytroph phenotype on minimal medium hinting at an alternative but less efficient pathway for production of phenylalanine exists in P. pastoris. Overall, all knockout vectors can easily be adapted to the gene of interest and strain background by efficient exchange of target homology regions and selection markers in single cloning steps. Average knockout efficiencies for all 25 genes were shown to be 40%, which is comparably high.