[Abstract unavailable

Mitotic yeast cells express five septins (Cdc3, Cdc10, Cdc11, Cdc12, and Shs1/Sep7). Only Shs1 is nonessential. The four essential septins form a complex containing two copies of each, but their arrangement was not known. Single-particle analysis by EM confirmed that the heterooligomer is octameric and revealed that the subunits are arrayed in a linear rod. Identity of each subunit was determined by examining complexes lacking a given septin, by antibody decoration, and by fusion to marker proteins (GFP or maltose binding protein). The rod has the order Cdc11-Cdc12-Cdc3-Cdc10 -Cdc10 -Cdc3-Cdc12-Cdc11 and, hence, lacks polarity. At low ionic strength, rods assemble end-to-end to form filaments but not when Cdc11 is absent or its N terminus is altered. Filaments invariably pair into long parallel ''railroad tracks.'' Lateral association seems to be mediated by heterotetrameric coiled coils between the paired C-terminal extensions of Cdc3 and Cdc12 projecting orthogonally from each filament. Shs1 may be able to replace Cdc11 at the end of the rod. Our findings provide insights into the molecular mechanisms underlying the function and regulation of cellular septin structures.electron microscopy ͉ yeast ͉ complexes ͉ GTP

No abstract

Signaling pathways that activate different mitogen-activated protein kinases (MAPKs) elicit many of the responses that are evoked in cells by changes in certain environmental conditions and upon exposure to a variety of hormonal and other stimuli. These pathways were first elucidated in the unicellular eukaryote Saccharomyces cerevisiae (budding yeast). Studies of MAPK pathways in this organism continue to be especially informative in revealing the molecular mechanisms by which MAPK cascades operate, propagate signals, modulate cellular processes, and are controlled by regulatory factors both internal to and external to the pathways. Here we highlight recent advances and new insights about MAPK-based signaling that have been made through studies in yeast, which provide lessons directly applicable to, and that enhance our understanding of, MAPK-mediated signaling in mammalian cells.

The Orm family proteins are conserved integral membrane proteins of the endoplasmic reticulum that are key homeostatic regulators of sphingolipid biosynthesis. Orm proteins bind to and inhibit serine: palmitoyl-coenzyme A transferase, the first enzyme in sphingolipid biosynthesis. In Saccharomyces cerevisiae, Orm1 and Orm2 are inactivated by phosphorylation in response to compromised sphingolipid synthesis (e.g., upon addition of inhibitor myriocin), thereby restoring sphingolipid production. We show here that protein kinase Ypk1, one of an essential pair of protein kinases, is responsible for this regulatory modification. Myriocin-induced hyperphosphorylation of Orm1 and Orm2 does not occur in ypk1Δ cells, and immunopurified Ypk1 phosphorylates Orm1 and Orm2 robustly in vitro exclusively on three residues that are known myriocin-induced sites. Furthermore, the temperature-sensitive growth of ypk1 ts ypk2Δ cells is substantially ameliorated by deletion of ORM genes, confirming that a primary physiological role of Ypk1-mediated phosphorylation is to negatively regulate Orm function. Ypk1 immunoprecipitated from myriocin-treated cells displays a higher specific activity for Orm phosphorylation than Ypk1 from untreated cells. To identify the mechanism underlying Ypk1 activation, we systematically tested several candidate factors and found that the target of rapamycin complex 2 (TORC2) kinase plays a key role. In agreement with prior evidence that a TORC2-dependent site in Ypk1(T662) is necessary for cells to exhibit a wild-type level of myriocin resistance, a Ypk1 (T662A) mutant displays only weak Orm phosphorylation in vivo and only weak activation in vitro in response to sphingolipid depletion. Additionally, sphingolipid depletion increases phosphorylation of Ypk1 at T662. Thus, Ypk1 is both a sensor and effector of sphingolipid level, and reduction in sphingolipids stimulates Ypk1, at least in part, via TORC2-dependent phosphorylation.cell regulation | plasma membrane

No abstract

Septins are a conserved family of GTP-binding proteins that assemble into symmetric linear hetero-oligomeric complexes, which, in turn, are able to polymerize into apolar filaments and higher-order structures. In budding yeast (Saccharomyces cerevisiae) and other eukaryotes, proper septin organization is essential for processes that involve membrane remodeling, such as the execution of cytokinesis. In yeast, four septin subunits form a Cdc11-Cdc12-Cdc3-Cdc10-Cdc10-Cdc3-Cdc12-Cdc11 hetero-octameric rod that polymerizes into filaments that are thought to form a collar around the bud neck in close contact with the inner surface of the plasma membrane. To explore septin-membrane interaction, we examined the effect of lipid monolayers on septin organization at the ultrastructural level using electron microcopy. Using this methodology we have acquired new insights concerning the potential effect of septin-membrane interactions on filament assembly, and more specifically on the role of phosphoinositides. Our studies demonstrate that budding yeast septins interact specifically with phosphatidylinositol-4,5-bisphosphate (PIP2) and indicate that the N-terminus of Cdc10 makes a major contribution to the interaction of septin filaments with PIP2. Furthermore, we found that presence of PIP2 promotes filament polymerization and organization on monolayers, even under conditions or for mutants that prevent filament formation in solution. In the extreme case of septin complexes lacking the normally terminal subunit Cdc11, or the normally central Cdc10 doublet, the combination of the PIP2-containing monolayer and nucleotide permitted filament formation in vitro via atypical Cdc12-Cdc12 and Cdc3-Cdc3 interactions, respectively.

All cells have the capacity to evoke appropriate and measured responses to signal molecules (such as peptide hormones), environmental changes, and other external stimuli. Tremendous progress has been made in identifying the proteins that mediate cellular response to such signals and in elucidating how events at the cell surface are linked to subsequent biochemical changes in the cytoplasm and nucleus. An emerging area of investigation concerns how signaling components are assembled and regulated (both spatially and temporally), so as to control properly the specificity and intensity of a given signaling pathway. A related question under intensive study is how the action of an individual signaling pathway is integrated with (or insulated from) other pathways to constitute larger networks that control overall cell behavior appropriately. This review describes the signal transduction pathway used by budding yeast (Saccharomyces cerevisiae) to respond to its peptide mating pheromones. This pathway is comprised by receptors, a heterotrimeric G protein, and a protein kinase cascade all remarkably similar to counterparts in multicellular organisms. The primary focus of this review, however, is recent advances that have been made, using primarily genetic methods, in identifying molecules responsible for regulation of the action of the components of this signaling pathway. Just as many of the constituent proteins of this pathway and their interrelationships were first identified in yeast, the functions of some of these regulators have clearly been conserved in metazoans, and others will likely serve as additional models for molecules that carry out analogous roles in higher organisms.