Abstract
Signalling is a key feature of living cells which frequently involves the local clustering of specific proteins in the plasma membrane. How such protein clustering is achieved within membrane microdomains (“rafts”) is an important, yet largely unsolved problem in cell biology. The plasma membrane of yeast cells represents a good model to address this issue, since it features protein domains that are sufficiently large and stable to be observed by fluorescence microscopy. Here, we demonstrate the ability of single-molecule atomic force microscopy to resolve lateral clustering of the cell integrity sensor Wsc1 in living Saccharomyces cerevisiae cells. We first localize individual wild-type sensors on the cell surface, revealing that they form clusters of ∼200 nm size. Analyses of three different mutants indicate that the cysteine-rich domain of Wsc1 has a crucial, not yet anticipated function in sensor clustering and signalling. Clustering of Wsc1 is strongly enhanced in deionized water or at elevated temperature, suggesting its relevance in proper stress response. Using in vivo GFP-localization, we also find that non-clustering mutant sensors accumulate in the vacuole, indicating that clustering may prevent endocytosis and sensor turnover. This study represents the first in vivo single-molecule demonstration for clustering of a transmembrane protein in S. cerevisiae. Our findings indicate that in yeast, like in higher eukaryotes, signalling is coupled to the localized enrichment of sensors and receptors within membrane patches.
Highlights
The evolution of uni- and multicellular organisms has produced a variety of cellular devices which enable cells to react to environmental changes
A prominent example are signalling processes, which in very diverse biological systems, from chemotaxis in E. coli [25] to the immune response in human T lymphocytes [26], frequently rely on - or are at least enhanced by the local clustering of signalling components in or near microdomains of the plasma membrane
Protein domains in yeast membranes have been shown to be sufficiently large and stable to be resolved by fluorescence microscopy [7]
Summary
The evolution of uni- and multicellular organisms has produced a variety of cellular devices which enable cells to react to environmental changes. The lateral organization of protein complexes within the plasma membrane in specific microdomains enriched in sphingolipids and sterols (‘‘lipid rafts’’) [3] is thought to be crucial in a variety of signalling processes governing diverse biological reactions such as endocytosis, cell adhesion, apoptosis or immune responses (reviewed in [4]). In the yeast Saccharomyces cerevisiae, the unicellular model eukaryote, stable microdomains have been observed within the plasma membrane by the use of specific GFPlabelled marker proteins [7] These fluorescence studies have shown that a so-called MCP (for ‘‘membrane compartment with Pma1’’) compartment forms a network-like structure, defined by its constituent proton ATPase Pma, while the MCC (‘‘membrane compartment with Can1’’) compartment forms 300 nm patches and houses a number of proton symporters, as well as a component of the eisosomes [8]
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