title: Single-molecule atomic force microscopy reveals clustering of the yeast plasma-membrane sensor Wsc1.
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 approximately 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.
authors.affiliation: Fachbereich Biologie/Chemie - AG Genetik, Universität Osnabrück, Osnabrück, Germany. Juergen.Heinisch@Biologie.Uni-Osnabrueck.DE
authors.forename: Jürgen J
authors.initials: JJ
authors.lastname: Heinisch
authors.forename: Vincent
authors.initials: V
authors.lastname: Dupres
authors.forename: Sabrina
authors.initials: S
authors.lastname: Wilk
authors.forename: Arne
authors.initials: A
authors.lastname: Jendretzki
authors.forename: Yves F
authors.initials: YF
authors.lastname: Dufrêne
mesh_terms: D000595:Amino Acid Sequence; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D018625:Microscopy, Atomic Force / Q000379:methods*; D008969:Molecular Sequence Data; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid
chemical_list: D008565:Membrane Proteins; C119383:SLG1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins