Inducible degradation-coupled phosphoproteomics identifies PP2ARts1 as a novel eisosome regulator.

Introduction: Reversible protein phosphorylation is an abundant post-translational modification dynamically regulated by opposing kinases and phosphatases. Protein phosphorylation has been extensively studied in cell division, where waves of cyclin-dependent kinase activity, peaking in mitosis, drive the sequential stages of the cell cycle. Here we developed and employed a strategy to specifically probe kinase or phosphatase substrates at desired times or experimental conditions in the model organism Saccharomyces cerevisiae. Methods: We combined auxin-inducible degradation (AID) with mass spectrometry-based phosphoproteomics, which allowed us to arrest physiologically normal cultures in mitosis prior to rapid phosphatase degradation and phosphoproteome analysis. Results and discussion: Our results revealed that protein phosphatase 2A coupled with its B56 regulatory subunit, Rts1 (PP2ARts1), is involved in dephosphorylation of numerous proteins in mitosis, highlighting the need for phosphatases to selectively maintain certain proteins in a hypophosphorylated state in the face of high mitotic kinase activity. Unexpectedly, we observed elevated phosphorylation at many sites on several subunits of the fungal eisosome complex following rapid Rts1 degradation. Eisosomes are dynamic polymeric assemblies that create furrows in the plasma membrane important in regulating nutrient import, lipid metabolism, and stress responses, among other things. We found that PP2ARts1-mediated dephosphorylation of eisosomes promotes their plasma membrane association and we provide evidence that this regulation impacts eisosome roles in metabolic homeostasis. The combination of rapid, inducible protein degradation with proteomic profiling offers several advantages over common protein disruption methods for characterizing substrates of regulatory enzymes involved in dynamic biological processes.

Ferroptosis-protective membrane domains in quiescence.

Quiescence is a common cellular state, required for stem cell maintenance and microorganismal survival under stress conditions or starvation. However, the mechanisms promoting quiescence maintenance remain poorly known. Plasma membrane components segregate into distinct microdomains, yet the role of this compartmentalization in quiescence remains unexplored. Here, we show that flavodoxin-like proteins (FLPs), ubiquinone reductases of the yeast eisosome membrane compartment, protect quiescent cells from lipid peroxidation and ferroptosis. Eisosomes and FLPs expand specifically in respiratory-active quiescent cells, and mutants lacking either show accelerated aging and defective quiescence maintenance and accumulate peroxidized phospholipids with monounsaturated or polyunsaturated fatty acids (PUFAs). FLPs are essential for the extramitochondrial regeneration of the lipophilic antioxidant ubiquinol. FLPs, alongside the Gpx1/2/3 glutathione peroxidases, prevent iron-driven, PUFA-dependent ferroptotic cell death. Our work describes ferroptosis-protective mechanisms in yeast and introduces plasma membrane compartmentalization as an important factor in the long-term survival of quiescent cells.

Inducible degradation-coupled phosphoproteomics identifies PP2ARts1 as a novel eisosome regulator.

Reversible protein phosphorylation is an abundant post-translational modification dynamically regulated by opposing kinases and phosphatases. Protein phosphorylation has been extensively studied in cell division, where waves of cyclin-dependent kinase activity, peaking in mitosis, drive the sequential stages of the cell cycle. Here we developed and employed a strategy to specifically probe kinase or phosphatase substrates at desired times or experimental conditions in the model organism Saccharomyces cerevisiae. We combined auxin-inducible degradation (AID) with mass spectrometry-based phosphoproteomics, which allowed us to arrest physiologically normal cultures in mitosis prior to rapid phosphatase degradation and phosphoproteome analysis. Our results revealed that protein phosphatase 2A coupled with its B56 regulatory subunit, Rts1 (PP2ARts1), is involved in dephosphorylation of numerous proteins in mitosis, highlighting the need for phosphatases to selectively maintain certain proteins in a hypophosphorylated state in the face of high mitotic kinase activity. Unexpectedly, we observed elevated phosphorylation at many sites on several subunits of the fungal eisosome complex following rapid Rts1 degradation. Eisosomes are dynamic polymeric assemblies that create furrows in the plasma membrane important in regulating nutrient import, lipid metabolism, and stress responses, among other things. We found that PP2ARts1-mediated dephosphorylation of eisosomes promotes their plasma membrane association and we provide evidence that this regulation impacts eisosome roles in metabolic homeostasis. The combination of rapid, inducible protein degradation with proteomic profiling offers several advantages over common protein disruption methods for characterizing substrates of regulatory enzymes involved in dynamic biological processes.

Phosphatidylinositol 3-phosphate regulates iron transport via PI3P-binding CgPil1 protein.

Iron homeostasis, which is pivotal to virulence, is regulated by the phosphatidylinositol 3-kinase CgVps34 in the human fungal pathogen Candida glabrata. Here, we identify CgPil1 as a phosphatidylinositol 3-phosphate (PI3P)-binding protein and unveil its role in retaining the high-affinity iron transporter CgFtr1 at the plasma membrane (PM), with PI3P negatively regulating CgFtr1-CgPil1 interaction. PI3P production and its PM localization are elevated in the high-iron environment. Surplus iron also leads to intracellular distribution and vacuolar delivery of CgPil1 and CgFtr1, respectively, from the PM. Loss of CgPil1 or CgFtr1 ubiquitination at lysines 391 and 401 results in CgFtr1 trafficking to the endoplasmic reticulum and a decrease in vacuole-localized CgFtr1. The E3-ubiquitin ligase CgRsp5 interacts with CgFtr1 and forms distinct CgRsp5-CgFtr1 puncta at the PM, with high iron resulting in their internalization. Finally, PI3P controls retrograde transport of many PM proteins. Altogether, we establish PI3P as a key regulator of membrane transport in C. glabrata.

The cytoplasmic tail of the mechanosensitive channel Pkd2 regulates its internalization and clustering in eisosomes.

Polycystins are a family of conserved ion channels, mutations of which lead to one of the most common human genetic disorders, namely, autosomal dominant polycystic kidney disease. Schizosacchromyces pombe possesses an essential polycystin homologue, Pkd2, which directs Ca2+ influx on the cell surface in response to membrane tension, but its structure remains unsolved. Here, we analyzed the structure-function relationship of Pkd2 based on its AlphaFold-predicted structure. Pkd2 consists of three domains, the extracellular lipid-binding domain (LBD), nine-helix transmembrane domain (TMD) and C-terminal cytoplasmic domain (CCD). Our genetic and microscopy data revealed that LBD and TMD are essential for targeting Pkd2 to the plasma membrane from the endoplasmic reticulum. In comparison, CCD ensures the polarized distribution of Pkd2 by promoting its internalization and preventing its clustering in the eisosome, a caveolae-like membrane compartment. The domains of Pkd2 and their functions are conserved in other fission yeast species. We conclude that both extracellular and cytoplasmic domains of Pkd2 are crucial for its intracellular trafficking and function. We propose that mechanosensitive channels can be desensitized through either internalization or clustering in low-tension membrane compartments.

Complementation of an Eisosomal Yeast pil1 Mutant and Characteristics of Eisosomal Distribution in Hyphae of Neurospora crassa Germinating from Two Different Spore Types.

Eisosomes are plasma-membrane-associated protein complexes of fungi and algae involved in various cellular processes. The eisosome composition of the budding yeast is well described, but there is a limited number of studies only about eisosomes in filamentous fungi. In our study, we examined the Neurospora crassa LSP-1 protein (NcLSP1). By complementing a Saccharomyces cerevisiae Δpil1 mutant strain with nclsp1, we show the functional homology of the NcLSP1 to yeast PIL1 rather than to yeast LSP1 and hereby confirm that the NcLSP1 is an eisosomal core protein and suitable eisosomal marker. The subsequent cloning and expression of the nclsp1::trfp reporter gene construct in N. crassa allowed for a systematical investigation of the characteristics of eisosome formation and distribution in different developmental stages. In N. crassa, the hyphae germinating from sexual and asexual spores are morphologically identical and have been historically recognized as the same type of cells. Here, we demonstrate the structural differences on the cellular level between the hyphae germinating from sexual and asexual spores.

Lsp1 partially substitutes for Pil1 function in eisosome assembly under stress conditions.

Eisosomes are large hemitubular structures that underlie the invaginated microdomains in the plasma membrane of various ascomycetous fungi, lichens and unicellular algae. In fungi, they are organized by BAR-domain containing proteins of the Pil1 family. Two such proteins, Pil1 and Lsp1, participate in eisosome formation in the yeast Saccharomyces cerevisiae. Under normal laboratory conditions, deletion of the PIL1 gene results in the inability of cells to assemble wild-type-like eisosomes. We found that under certain stress conditions, Lsp1 partially substitutes for the Pil1 function and mediates assembly of eisosomes, specifically following a decrease in the activity of serine palmitoyltransferase, for example, in response to hyperosmotic stress. Besides Lsp1, the assembly of eisosomes lacking Pil1 also requires Seg1 and Nce102 proteins. Using next-generation sequencing, we found that the seg1Δnce102Δpil1Δ strain, which is unable to form eisosomes, overexpresses genes coding for proteins of oxidative phosphorylation and tricarboxylic acid cycle. By contrast, genes involved in DNA repair, ribosome biogenesis and cell cycle are downregulated. Our results identify Lsp1 as a stress-responsive eisosome organizer and indicate several novel functional connections between the eisosome and essential cellular processes.

The eisosomes contribute to acid tolerance of yeast by maintaining cell membrane integrity.

Microbes have evolved multiple mechanisms to resist environmental stresses, which are regulated in complex and delicate ways. Though the role of cell membranes in acid resistance from the perspective of physicochemical properties and membrane proteins has been deeply studied, the function of eisosomes is still in its infancy. In this study, we firstly reported the dynamic changes of eisosomes under acid stress and the decreased acid tolerance of yeasts caused by eisosome disruption. Physiological indicators and non-targeted lipid profiling revealed that eisosome disruption caused changes in multiple lipids and imbalances in lipid homeostasis, which are responsible for membrane integrity damage. Thus the increased infiltration of carboxylic acids and the raised ROS levels were detected in strains with disrupted eisosome assembly, resulting in decreased cellular tolerance. The results here provide novel insights into the acid-resistant mechanism of yeasts from the perspective of the cell membrane subdomain, which has practical impacts on green biological manufacturing and food preservation.

Microdomain Protein Nce102 Is a Local Sensor of Plasma Membrane Sphingolipid Balance.

Sphingolipids are essential building blocks of eukaryotic membranes and important signaling molecules that are regulated tightly in response to environmental and physiological inputs. While their biosynthetic pathway has been well-described, the mechanisms that facilitate the perception of sphingolipid levels at the plasma membrane remain to be uncovered. In Saccharomyces cerevisiae, the Nce102 protein has been proposed to function as a sphingolipid sensor as it changes its plasma membrane distribution in response to sphingolipid biosynthesis inhibition. We show that Nce102 redistributes specifically in regions of increased sphingolipid demand, e.g., membranes of nascent buds. Furthermore, we report that the production of Nce102 increases following sphingolipid biosynthesis inhibition and that Nce102 is internalized when excess sphingolipid precursors are supplied. This finding suggests that the total amount of Nce102 in the plasma membrane is a measure of the current need for sphingolipids, whereas its local distribution marks sites of high sphingolipid demand. The physiological role of Nce102 in the regulation of sphingolipid synthesis is demonstrated by mass spectrometry analysis showing reduced levels of hydroxylated complex sphingolipids in response to heat stress in the nce102Δ deletion mutant. We also demonstrate that Nce102 behaves analogously in the widespread human fungal pathogen Candida albicans, suggesting a conserved principle of local sphingolipid control across species. IMPORTANCE Microorganisms are challenged constantly by their rapidly changing environment. To survive, they have developed diverse mechanisms to quickly perceive stressful situations and adapt to them appropriately. The primary site of both stress sensing and adaptation is the plasma membrane. We identified the yeast protein Nce102 as a marker of local sphingolipid levels and fluidity in the plasma membrane. Nce102 is an important structural and functional component of the membrane compartment Can1 (MCC), a plasma membrane microdomain stabilized by a large cytosolic hemitubular protein scaffold, the eisosome. The MCC/eisosomes are widely conserved among fungi and unicellular algae. To determine if Nce102 carries out similar functions in other organisms, we analyzed the human fungal pathogen Candida albicans and found that Nce102 responds to sphingolipid levels also in this organism, which has potential applications for the development of novel therapeutic approaches. The presented study represents a valuable model for how organisms regulate plasma membrane sphingolipids.

Coordinated cortical ER remodeling facilitates actomyosin ring assembly.

The cortical endoplasmic reticulum (cER) is a reticulated network closely attached to the plasma membrane (PM). In the fission yeast Schizosaccharomyces pombe (S. pombe), ER-PM contacts have been suggested to restrict both the allocation and compaction of large-sized actomyosin assemblies along the lateral cell cortex. However, how cells orchestrate ER-PM contact remodeling in accordance with actomyosin coalescence for contractile ring assembly is unclear. Here, we reveal that actomyosin compaction directs the remodeling of the free tubular cER edges, whereas active exocytosis subsequently promotes the reorganization of the eisosome-bound cER rims by weakening their association or repatterning the eisosome-coated PM furrows. cER-eisosome contacts also act to reserve tubular cER edges and, hence, the ER shaping machinery at the lateral cell cortex. By manipulating or rerouting exocytosis in mutants with compromised actomyosin compaction, due to either the loss of myosin II activity or sheet-like cER morphology, we show that exocytosis facilitates ring formation likely by creating free tubular cER rims allowing robust cER remodeling. We thus propose that coordinated cER remodeling driven by both actomyosin forces and active exocytosis ensures proper contractile ring assembly. Our work also provides mechanistic insights into cER-related modulation in actomyosin ring assembly.

Coordinated regulation of TORC2 signaling by MCC/eisosome-associated proteins, Pil1 and tetraspan membrane proteins during the stress response.

MCCs are linear invaginations of the yeast plasma membrane that form stable membrane microdomains. Although over 20 proteins are localized in the MCCs, it is not well understood how these proteins coordinately maintain normal MCC function. Pil1 is a core eisosome protein and is responsible for MCC-invaginated structures. In addition, six-tetraspan membrane proteins (6-Tsp) are localized in the MCCs and classified into two families, the Sur7 family and Nce102 family. To understand the coordinated function of these MCC proteins, single and multiple deletion mutants of Pil1 and 6-Tsp were generated and their MCC structure and growth under various stresses were investigated. Genetic interaction analysis revealed that the Sur7 family and Nce102 function in stress tolerance and normal eisosome assembly, respectively, by cooperating with Pil1. To further understand the role of MCCs/eisosomes in stress tolerance, we screened for suppressor mutants using the SDS-sensitive phenotype of pil1Δ 6-tspΔ cells. This revealed that SDS sensitivity is caused by hyperactivation of Tor kinase complex 2 (TORC2)-Ypk1 signaling. Interestingly, inhibition of sphingolipid metabolism, a well-known downstream pathway of TORC2-Ypk1 signaling, did not rescue the SDS-sensitivity of pil1Δ 6-tspΔ cells. These results suggest that Pil1 and 6-Tsp cooperatively regulate TORC2 signaling during the stress response.

Eisosome disruption by noncoding RNA deletion increases protein secretion in yeast.

Noncoding RNAs (ncRNAs) regulate many aspects of gene expression. We investigated how ncRNAs affected protein secretion in yeast by large-scale screening for improved endogenous invertase secretion in ncRNA deletion strains with deletion of stable unannotated transcripts (SUTs), cryptic unstable transcripts (CUTs), tRNAs, or snRNAs. We identified three candidate ncRNAs, SUT418, SUT390, and SUT125, that improved endogenous invertase secretion when deleted. As SUTs can affect expression of nearby genes, we quantified adjacent gene transcription and found that the PIL1 gene was down-regulated in the SUT125 deletion strain. Pil1 is a core component of eisosomes, nonmobile invaginations found throughout the plasma membrane. PIL1 knockout alone, or in combination with eisosome components LSP1 or SUR7, resulted in further increased secretion of invertase. Secretion of heterologous GFP was also increased upon PIL1 deletion, but this increase was signal sequence dependent. To reveal the potential for increased biopharmaceutical production, secretion of monoclonal antibody Pexelizumab scFv peptide was increased by PIL1 deletion. Global analysis of secreted proteins revealed that approximately 20% of secreted proteins, especially serine-enriched secreted proteins, including invertase, were increased upon eisosome disruption. Eisosomes are enriched with APC transporters and sphingolipids, which are essential components for secretory vesicle formation and protein sorting. Sphingolipid and serine biosynthesis pathways were up-regulated upon PIL1 deletion. We propose that increased secretion of endogenous and heterologous proteins upon PIL1 deletion resulted from sphingolipid redistribution in the plasma membrane and up-regulated sphingolipid biosynthesis. Overall, a new pathway to improve protein secretion in yeast via eisosome disruption has been identified.

A virulence-related lectin traffics into eisosome and contributes to functionality of cytomembrane and cell-wall in the insect-pathogenic fungus Beauveria bassiana.

Lectins are characterized of the carbohydrate-binding ability and play comprehensive roles in fungal physiology (e.g., defense response, development and host-pathogen interaction). Beauveria bassiana, a filamentous entomopathogenic fungus, has a lectin-like protein containing a Fruit Body_domain (BbLec1). BbLec1 could bind to chitobiose and chitin in fungal cell wall. BbLec1 proteins interacted with each other to form multimers, and translocated into eisosomes. Further, the interdependence between BbLec1 and the eisosome protein PliA was essential for stabilizing the eisosome architecture. To test the BbLec1 roles in B. bassiana, we constructed the gene disruption and complementation mutants. Notably, the BbLec1 loss resulted in the impaired cell wall in mycelia and conidia as well as conidial formation capacity. In addition, disruption of BbLec1 led to the reduced cytomembrane integrity and the enhanced sensitivity to osmotic stress. Finally, ΔBbLec1 mutant strain displayed the weakened virulence when compared with the wild-type strain. Taken together, BbLec1 traffics into eisosome and links the functionality of eisosome to development and virulence of B. bassiana.

The Sur7 cytoplasmic C terminus regulates morphogenesis and stress responses in Candida albicans.

MCC/eisosome subdomains of the plasma membrane promote proper cell wall morphogenesis that is critical for the fungal pathogen Candida albicans to grow invasively and resist stressful environments in the host. Sur7 localizes to MCC/eisosomes and is needed for their function, so in this work, the role of this tetraspan membrane protein was studied by mutagenesis. Deletion mutant analysis showed that the N-terminal region containing the four transmembrane domains mediates Sur7 localization to MCC/eisosomes. Mutation of 32 conserved residues in the N-terminal region indicated that extracellular loop 1 is important, although these mutants generally displayed weak phenotypes. Surprisingly, two Cys residues in a conserved motif in extracellular loop 1 were not important. However, deletion of the entire 15 amino acid motif revealed that it was needed for proper membrane trafficking of Sur7. Deletion and substitution mutagenesis showed that the C terminus is important for resisting cell wall stress. This is significant as it indicates Sur7 carries out an important role in the cytoplasm. Altogether, these results indicate that the N-terminal region localizes Sur7 to MCC/eisosomes and that the C-terminal domain promotes responses in the cytoplasm needed for cell wall morphogenesis and stress resistance.

Endocytosis of nutrient transporters in fungi: The ART of connecting signaling and trafficking.

Plasma membrane transporters play pivotal roles in the import of nutrients, including sugars, amino acids, nucleobases, carboxylic acids, and metal ions, that surround fungal cells. The selective removal of these transporters by endocytosis is one of the most important regulatory mechanisms that ensures a rapid adaptation of cells to the changing environment (e.g., nutrient fluctuations or different stresses). At the heart of this mechanism lies a network of proteins that includes the arrestin-related trafficking adaptors (ARTs) which link the ubiquitin ligase Rsp5 to nutrient transporters and endocytic factors. Transporter conformational changes, as well as dynamic interactions between its cytosolic termini/loops and with lipids of the plasma membrane, are also critical during the endocytic process. Here, we review the current knowledge and recent findings on the molecular mechanisms involved in nutrient transporter endocytosis, both in the budding yeast Saccharomyces cerevisiae and in some species of the filamentous fungus Aspergillus. We elaborate on the physiological importance of tightly regulated endocytosis for cellular fitness under dynamic conditions found in nature and highlight how further understanding and engineering of this process is essential to maximize titer, rate and yield (TRY)-values of engineered cell factories in industrial biotechnological processes.

Quick-Freeze, Deep-Etch Electron Microscopy Reveals the Characteristic Architecture of the Fission Yeast Spore.

The spore of the fission yeast Schizosaccharomyces pombe is a dormant cell that is resistant to a variety of environmental stresses. The S. pombe spore is coated by a proteinaceous surface layer, termed the Isp3 layer because it comprises mainly Isp3 protein. Although thin-section electron microscopy and scanning electron microscopy have revealed the fundamental structure of the spore, its architecture remains unclear. Here we visualized S. pombe spores by using a quick-freeze replica electron microscopy (QFDE-EM) at nanometer resolution, which revealed novel characteristic structures. QFDE-EM revealed that the Isp3 layer exists as an interwoven fibrillar layer. On the spore cell membrane, many deep invaginations, which are longer than those on the vegetative cell membrane, are aligned in parallel. We also observed that during spore germination, the cell surface changes from a smooth to a dendritic filamentous structure, the latter being characteristic of vegetative cells. These findings provide significant insight into not only the structural composition of the spore, but also the mechanism underlying the stress response of the cell.

Plasma Membrane Protein Nce102 Modulates Morphology and Function of the Yeast Vacuole.

Membrane proteins are targeted not only to specific membranes in the cell architecture, but also to distinct lateral microdomains within individual membranes to properly execute their biological functions. Yeast tetraspan protein Nce102 has been shown to migrate between such microdomains within the plasma membrane in response to an acute drop in sphingolipid levels. Combining microscopy and biochemistry methods, we show that upon gradual ageing of a yeast culture, when sphingolipid demand increases, Nce102 migrates from the plasma membrane to the vacuole. Instead of being targeted for degradation it localizes to V-ATPase-poor, i.e., ergosterol-enriched, domains of the vacuolar membrane, analogous to its plasma membrane localization. We discovered that, together with its homologue Fhn1, Nce102 modulates vacuolar morphology, dynamics, and physiology. Specifically, the fusing of vacuoles, accompanying a switch of fermenting yeast culture to respiration, is retarded in the strain missing both proteins. Furthermore, the absence of either causes an enlargement of ergosterol-rich vacuolar membrane domains, while the vacuoles themselves become smaller. Our results clearly show decreased stability of the V-ATPase in the absence of either Nce102 or Fhn1, a possible result of the disruption of normal microdomain morphology of the vacuolar membrane. Therefore, the functionality of the vacuole as a whole might be compromised in these cells.

The Sur7/PalI family transmembrane protein Tos7 (Yol019w) plays a role in secretion in budding yeast.

Tos7 (Yol019w) is a Sur7/PalI family transmembrane protein in the budding yeast Saccharomyces cerevisiae. Since the deletion of TOS7 did not affect growth or cell morphology, the cellular roles of Tos7 have not been established previously. Here, we show that high-copy TOS7 expression suppressed the growth defect of the secretion-defective RGA1-C term-overexpressing mutant and sec15-1 mutant. Moreover, Tos7 physically interacted with Boi2 and the Rho GTPase Rho3, two key regulators of exocyst assembly, suggesting that Tos7 plays a role in secretion. We also show that the deletion of TOS7 rendered the cells more sensitive to the cell wall-disrupting agents Congo red and calcofluor white while high-copy TOS7 expression had an opposite effect, suggesting that Tos7 affects cell wall organization. Finally, we show that Tos7 localized to punctate patches on the plasma membrane that were largely co-localized with the plasma membrane microdomains named MCC (membrane compartment of Can1). Together, these results suggest that Tos7 contributes to cell surface-related functions. Tos7 is likely an auxiliary component of MCC/eisosome that specifically interacts with the secretory pathway.

The Composition and the Structure of MCC/Eisosomes in Neurospora crassa.

MCC/eisosomes are protein-organized domains in the plasma membrane of fungi and algae. However, the composition and function(s) of MCC/eisosomes in the filamentous fungus Neurospora crassa were previously unknown. To identify proteins that localize to MCC/eisosomes in N. crassa, we isolated proteins that co-purified with the core MCC/eisosome protein LSP-1, which was tagged with GFP. Proteins that co-fractionated with LSP-1:GFP were then identified by mass spectrometry. Eighteen proteins were GFP-tagged and used to identify six proteins that highly colocalized with the MCC/eisosome marker LSP-1:RFP, while five other proteins showed partial overlap with MCC/eisosomes. Seven of these proteins showed amino acid sequence homology with proteins known to localize to MCC/eisosomes in the yeast Saccharomyces cerevisiae. However, homologs of three proteins known to localize to MCC/eisosomes in S. cerevisiae (Can1, Pkh1/2, and Fhn1) were not found to colocalize with MCC/eisosome proteins in N. crassa by fluorescence microscopy. Interestingly, one new eisosome protein (glutamine-fructose-6-phosphate aminotransferase, gene ID: NCU07366) was detected in our studies. These findings demonstrate that there are interspecies differences of the protein composition of MCC/eisosomes. To gain further insight, molecular modeling and bioinformatics analysis of the identified proteins were used to propose the organization of MCC/eisosomes in N. crassa. A model will be discussed for how the broad range of functions predicted for the proteins localized to MCC/eisosomes, including cell wall synthesis, response and signaling, transmembrane transport, and actin organization, suggests that MCC/eisosomes act as organizing centers in the plasma membrane.

Plasma Membrane MCC/Eisosome Domains Promote Stress Resistance in Fungi.

There is growing appreciation that the plasma membrane orchestrates a diverse array of functions by segregating different activities into specialized domains that vary in size, stability, and composition. Studies with the budding yeast Saccharomyces cerevisiae have identified a novel type of plasma membrane domain known as the MCC (membrane compartment of Can1)/eisosomes that correspond to stable furrows in the plasma membrane. MCC/eisosomes maintain proteins at the cell surface, such as nutrient transporters like the Can1 arginine symporter, by protecting them from endocytosis and degradation. Recent studies from several fungal species are now revealing new functional roles for MCC/eisosomes that enable cells to respond to a wide range of stressors, including changes in membrane tension, nutrition, cell wall integrity, oxidation, and copper toxicity. The different MCC/eisosome functions are often intertwined through the roles of these domains in lipid homeostasis, which is important for proper plasma membrane architecture and cell signaling. Therefore, this review will emphasize the emerging models that explain how MCC/eisosomes act as hubs to coordinate cellular responses to stress. The importance of MCC/eisosomes is underscored by their roles in virulence for fungal pathogens of plants, animals, and humans, which also highlights the potential of these domains to act as novel therapeutic targets.