Coupling of septins to the axial landmark by Bud4 in budding yeast.

Cells of the budding yeast Saccharomyces cerevisiae select a site for polarized growth in a specific pattern that depends on their cell type. Haploid a and α cells bud in the axial budding pattern, which requires assembly of a landmark that includes the Bud4 protein. To understand how an axial bud site is established, we performed a structure-function analysis of Bud4. Bud4 contains DUF1709 (domain of unknown function), which is similar to a part of the anillin-homology domain, and a putative Pleckstrin homology (PH) domain near to its C terminus. Although its localization depends on septins, a conserved family of GTP-binding proteins, Bud4 is necessary for the stable inheritance of septin rings during cell division. Although some anillins interact directly with septins, we find that neither DUF1709 nor the PH domain is necessary for targeting Bud4 to the mother-bud neck. Instead, this C-terminal region is crucial for association of Bud4 with Bud3 and other components of the axial landmark. Remarkably, septins colocalize with Bud4 mutant proteins that lack these C-terminal domains, forming an arc or a single ring instead of a double ring during and after cytokinesis. Interestingly, overexpression of Bud4 also induces formation of extra Bud4 rings and arcs that are associated with septins. Analyses of a series of bud4 truncation mutants suggest that at least two domains in the central region play a redundant role in targeting Bud4 to the mother-bud neck and are thus likely to interact with septins. Taken together, these results indicate that Bud4 functions as a platform that links septins to the axial landmark.

Identification of phosphatase 2A-like Sit4-mediated signalling and ubiquitin-dependent protein sorting as modulators of caffeine sensitivity in S. cerevisiae.

Caffeine exerts pleiotropic effects on eukaryotic cells via its ability to act as a low-affinity adenosine analogue. Here we report that the genes HSE1, RTS3, SDS23 and SDS24 confer caffeine resistance when overexpressed in S. cerevisiae. The Hse1 protein functions in ubiquitin-dependent vacuolar protein sorting, whereas the other proteins are poorly characterized. Bioinformatic analysis of genetic and physical interaction data linked Rts3 and Sds23/24 to the phosphatase 2A-like Sit4 pathway. Combinatorial deletions of the identified suppressor genes conferred varying levels of caffeine hypersensitivity. For hse1Δ and rts3Δ mutants, caffeine sensitivity was partially rescued by sorbitol osmostabilization, suggesting possible cell wall integrity defects in these strains. Rapamycin sensitivity experiments linked the caffeine sensitivity of rts3Δ, but not that of sds23/24Δ or hse1Δ strains, to inhibition of the TORC1 kinase complex, a central regulator of cell growth and a known caffeine target. Epistasis experiments support a model in which Rts3 and Sds23/24 act in parallel to negatively regulate Sit4, while Hse1 confers caffeine resistance via a separate pathway. In summary, this study identifies the Sit4 phosphatase pathway and membrane protein dynamics as key modulators of caffeine-mediated inhibition of yeast cell growth and proposes novel functions for Rts3 and Sds23/24.

TORC1 kinase and the S-phase cyclin Clb5 collaborate to promote mitotic spindle assembly and DNA replication in S. cerevisiae.

The Target of Rapamycin complex 1 (TORC1) is a central regulator of eukaryotic cell growth that is inhibited by the drug rapamycin. In the budding yeast Saccharomyces cerevisiae, translational defects associated with TORC1 inactivation inhibit cell cycle progression at an early stage in G1, but little is known about the possible roles for TORC1 later in the cell cycle. We investigated the rapamycin-hypersensitivity phenotype of cells lacking the S phase cyclin Clb5 (clb5Δ) as a basis for uncovering novel connections between TORC1 and the cell cycle regulatory machinery. Dosage suppression experiments suggested that the clb5Δ rapamycin hypersensitivity reflects a unique Clb5-associated cyclin-dependent kinase (CDK) function that cannot be performed by mitotic cyclins and that also involves motor proteins, particularly the kinesin-like protein Kip3. Synchronized cell experiments revealed rapamycin-induced defects in pre-anaphase spindle assembly and S phase progression that were more severe in clb5Δ than in wild-type cells but no apparent activation of Rad53-dependent checkpoint pathways. Some rapamycin-treated cells had aberrant spindle morphologies, but rapamycin did not cause gross defects in the microtubule cytoskeleton. We propose a model in which TORC1 and Clb5/CDK act coordinately to promote both spindle assembly via a pathway involving Kip3 and S phase progression.

A Western blot-based investigation of the yeast secretory pathway designed for an intermediate-level undergraduate cell biology laboratory.

The movement of newly synthesized proteins through the endomembrane system of eukaryotic cells, often referred to generally as the secretory pathway, is a topic covered in most intermediate-level undergraduate cell biology courses. An article previously published in this journal described a laboratory exercise in which yeast mutants defective in two distinct steps of protein secretion were differentiated using a genetic reporter designed specifically to identify defects in the first step of the pathway, the insertion of proteins into the endoplasmic reticulum (Vallen, 2002). We have developed two versions of a Western blotting assay that serves as a second way of distinguishing the two secretory mutants, which we pair with the genetic assay in a 3-wk laboratory module. A quiz administered before and after students participated in the lab activities revealed significant postlab gains in their understanding of the secretory pathway and experimental techniques used to study it. A second survey administered at the end of the lab module assessed student perceptions of the efficacy of the lab activities; the results of this survey indicated that the experiments were successful in meeting a set of educational goals defined by the instructor.

Cytoplasmic Clb2 is required for timely inactivation of the mitotic inhibitor Swe1 and normal bud morphogenesis in Saccharomyces cerevisiae.

Subcellular localization is an important determinant of substrate and functional specificity for cyclin-cyclin dependent kinase (CDK) complexes. This work addresses the cytoplasmic function of the budding yeast mitotic cyclin Clb2, which is mostly nuclear but is also present in the bulk cytoplasm and at the mother-bud neck. Clb2 contains two leucine-rich nuclear export signals (NESs)--one of which we newly describe here--that maintain its presence in the cytoplasm. Yeast strains bearing mutations in one or both of these NESs have elongated buds, indicative of a G2/M cell cycle delay. A small number of these cells exhibit a filamentous-like morphology under conditions that do not normally induce filamentous growth. These phenotypes are enhanced by deletion of the other three mitotic cyclins (CLB1,3,4) and are dependent on expression of Swe1, the yeast Cdk1 inhibitory kinase. Deltaclb1,3,4 Deltabud3 cells, which fail to localize Clb2 to the bud neck, also exhibit a Swe1-dependent elongated bud phenotype. Our results support a model in which cytoplasmic Clb2-Cdk1 is required for timely inactivation of Swe1 at the G2/M transition and bud neck targeting of Clb2 contributes to the efficiency of this process. Cytoplasmic Clb2 may also be important for repression of filamentous growth.