Highlights d In yeast, some cell-cycle activators increase in concentration as cells grow larger d Cell-cycle inhibitors decrease in concentration as cells grow larger d These effects can explain why cells divide at increased sizes d Reversal of this natural scaling of expression caused poor cell size control

One of the best characterized fungal membrane microdomains is the MCC/eisosome. The MCC (membrane compartment of Can1) is an evolutionarily conserved ergosterol-rich plasma membrane domain. It is stabilized on its cytosolic face by the eisosome, a hemitubular protein complex composed of Bin/Amphiphysin/Rvs (BAR) domain-containing Pil1 and Lsp1. These two proteins bind directly to phosphatidylinositol 4,5-bisphosphate and promote the typical furrow-like shape of the microdomain, with highly curved edges and bottom. While some proteins display stable localization in the MCC/eisosome, others enter or leave it under particular conditions, such as misbalance in membrane lipid composition, changes in membrane tension, or availability of specific nutrients. These findings reveal that the MCC/eisosome, a plasma membrane microdomain with distinct morphology and lipid composition, acts as a multifaceted regulator of various cellular processes including metabolic pathways, cellular morphogenesis, signalling cascades, and mRNA decay. In this minireview, we focus on the MCC/eisosome’s proposed role in the regulation of lipid metabolism. While the molecular mechanisms of the MCC/eisosome function are not completely understood, the idea of intracellular processes being regulated at the plasma membrane, the foremost barrier exposed to environmental challenges, is truly exciting.

We report the transmembrane voltage-induced lateral reorganization of highly-ordered lipid microdomains in the plasma membrane of living Saccharomyces cerevisiae. Using trans-parinaric acid (all-trans-9,11,13,15-octadecatetraenoic acid) as a probe of lipid order and different methods of membrane depolarization, we found that depolarization always invokes a significant reduction in the amount of gel-like microdomains in the membrane. Different depolarization mechanisms, including the application of ionophores, cell depolarization by an external electric field, depolarization by proton/hexose co-transport facilitated by HUP1 protein and a reduction of membrane potential caused by compromised respiration efficiency, yielded the same results independently of the yeast strain used. The data suggest that the voltage-induced reorganization of lateral membrane structure could play significant role in fast cellular response to acute stress conditions, as well as in other membrane microdomain-related regulatory mechanisms.

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.

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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.

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 the Pil1 function and assembles eisosomes, specifically following a decrease in the activity of serine-palmitoyl transferase, e.g., 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. On the other hand, 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.

Tok1p is a highly specific yeast plasma membrane potassium channel with strong outward directionality. Its opening is induced by membrane depolarization. Although the biophysical properties of Tok1p are well-described, its potentially important physiological role is currently largely unexplored. To address this issue, we examined the Tok1p activity following chemically-induced depolarization by measuring changes of plasma membrane potential (ΔΨ) using the diS-C(3) fluorescence assay in a Tok1p-expressing and a Tok1p-deficient strain. We report that Tok1p channel activity in response to chemical stress does not depend solely on the extent of depolarization, as might have been expected, but may also be negatively influenced by accompanying effects of the used compound. The stressors may interact with the plasma membrane or the channel itself, or cause cytosolic acidification. All of these effects may negatively influence the Tok1p channel opening. While ODDC-induced depolarization exhibits the cleanest Tok1p activation, restoring an astonishing 75% of lost ΔΨ, higher BAC concentrations reduce Tok1p activity, probably because of direct interactions with the channel and/or its lipid microenvironment. This is not only the first study of the physiological role of Tok1p in ΔΨ maintenance under chemical stress, but also the first estimate of the extent of depolarization the channel is able to counterbalance.