Bettina Zanolari

Sphingoid base synthesis requirement for endocytosis in Saccharomyces cerevisiae.

The internalization step of endocytosis in yeast requires actin and sterols for maximum efficiency. In addition, many receptors and plasma membrane proteins must be phosphorylated and ubiquitylated prior to internalization. The Saccharomyces cerevisiae end8‐1 mutant is allelic to lcb1, a mutant defective in the first step of sphingoid base synthesis. Upon arrest of sphingoid base synthesis a rapid block in endocytosis is seen. This block can be overcome by exogenous sphingoid base. Under conditions where endogenous sphingosine base synthesis was blocked and exogenous sphingoid bases could not be converted to phosphorylated sphingoid bases or to ceramide, sphingoid bases could still suppress the endocytic defect. Therefore, the required lipid is most likely a sphingoid base. Interestingly, sphingoid base synthesis is required for proper actin organization, but is not required for receptor phosphorylation. This is the first case of a physiological role for sphingoid base synthesis, other than as a precursor for ceramide or phosphorylated sphingoid base synthesis.

Increased protein kinase or decreased PP2A activity bypasses sphingoid base requirement in endocytosis.

Lipids have been implicated in signal transduction and in several stages of membrane trafficking, but these two functions have not been functionally linked. In yeast, sphingoid base synthesis is required for the internalization step of endocytosis and organization of the actin cytoskeleton. We show that inactivation of a protein phosphatase 2A (PP2A) or overexpression of one of two kinases, Yck2p or Pkc1p, can specifically suppress the sphingoid base synthesis requirement for endocytosis. The two kinases have an overlapping function because only a mutant with impaired function of both kinases is defective in endocytosis. An ultimate target of sphingoid base synthesis may be the actin cytoskeleton, because overexpression of the kinases and inactivation of PP2A substantially corrected the actin defect due to the absence of sphingoid base. These results suggest that sphingoid base controls protein phosphorylation, perhaps by activating a signal transduction pathway that is required for endocytosis and proper actin cytoskeleton organization in yeast.

Yeast pheromone receptor endocytosis and hyperphosphorylation are independent of G protein-mediated signal transduction

When alpha factor binds to the yeast alpha factor receptor a signal is transmitted via a tripartite G protein that prepares the cell for conjugation. As a result of alpha factor binding the receptor also undergoes a regulated internalization and hyperphosphorylation. Using cells that lack activity of this tripartite G protein, we show that G protein-mediated pheromone signal transduction is not necessary for regulation of receptor internalization or hyperphosphorylation. Therefore, the processes of signal transduction and down regulation can be uncoupled. We propose that binding of alpha factor to its receptor results in a receptor conformation change that permits receptor hyperphosphorylation and interaction with the endocytic machinery.

Transport to the plasma membrane is regulated differently early and late in the cell cycle in Saccharomyces cerevisiae

Traffic from the trans-Golgi network to the plasma membrane is thought to occur through at least two different independent pathways. The chitin synthase Chs3p requires the exomer complex and Arf1p to reach the bud neck of yeast cells in a cell-cycle-dependent manner, whereas the hexose transporter Hxt2p localizes over the entire plasma membrane independently of the exomer complex. Here, we conducted a visual screen for communalities and differences between the exomer-dependent and exomer-independent transport to the plasma membrane in Saccharomyces cerevisiae. We found that most of the components that are required for the fusion of transport vesicles with the plasma membrane, are involved in localization of both Chs3p and Hxt2p. However, the lethal giant larva homologue Sro7p is required primarily for the targeting of Chs3p, and not Hxt2p or other cargoes such as Itr1p, Cwp2p and Pma1p. Interestingly, this transport defect was more pronounced in large-budded cells just before cytokinesis than in small-budded cells. In addition, we found that the yeast Rab11 homologue Ypt31p determines the residence time of Chs3p in the bud neck of small-budded, but not large-budded, cells. We propose that transport to and from the bud neck is regulated differently in small- and large-budded cells, and differs early and late in the cell cycle.

Quantitation of alpha-factor internalization and response during the Saccharomyces cerevisiae cell cycle.

The alpha-factor pheromone binds to specific cell surface receptors on Saccharomyces cerevisiae a cells. The pheromone is then internalized, and cell surface receptors are down-regulated. At the same time, a signal is transmitted that causes changes in gene expression and cell cycle arrest. We show that the ability of cells to internalize alpha-factor is constant throughout the cell cycle, a cells are also able to respond to pheromone throughout the cycle even though there is cell cycle modulation of the expression of two pheromone-inducible genes, FUS1 and STE2. Both of these genes are expressed less efficiently near or just after the START point of the cell cycle in response to alpha-factor. For STE2, the basal level of expression is modulated in the same manner.

Involvement of the exomer complex in the polarized transport of Ena1 required for Saccharomyces cerevisiae survival against toxic cations

Here we show that the TGN complex named exomer is required for alkali cation tolerance in yeast because of its roles in the sorting and polarization of the plasma membrane Na+-ATPase Ena1 and on the signal processing through the RIM101 pathway, thus widening the functional repertoire of the yeast exomer.

Receptor-Mediated Endocytosis of α-Factor by S. Cerevisiae a Cells

S. cerevisiae exists in two haploid cell types, a and α, which mate with each other to form the diploid a/α cell. Essential to the mating process is the reciprocal action of two peptide mating factors called a- and α-factor. The factors induce changes in the pattern of mRNA and protein synthesis, an arrest in the G1 phase of the cell cycle and a morphological change called the “shmoo”. This oriented projection is the site where the two haploid cells fuse (see Cross et al 1988 for a review). The a- and α-factor receptors have been identified as the products of the STE3 and STE2 genes respectively (Jenness et al 1983; Nakayama et al 1985; Hagen et al 1986). These two receptors are polytopic membrane proteins that share a common basic structure with the β- adrenergic receptor (Dixon et al 1986). Like this receptor, the STE2 and STE3 gene products are coupled to a G protein (Dietzel and Kurjan 1987; Miyajima et al 1987; Whiteway et al 1989). The enzyme controlled by the G-protein has not been identified although several other genes acting downstream in the signal transduction pathway are known (Nakayama et al 1988; Blinder et al 1989).