Relatively little is known about the in vivo function of individual components of the eukaryotic γ-tubulin complex (γ-TuC). We identified three genes, gfh1+, mod21+, and mod22+, in a screen for fission yeast mutants affecting microtubule organization. gfh1+ is a previously characterized γ-TuC protein weakly similar to human γ-TuC subunit GCP4, whereas mod21+ is novel and shows weak similarity to human γ-TuC subunit GCP5. We show that mod21p is a bona fide γ-TuC protein and that, like gfh1Δ mutants, mod21Δ mutants are viable. We find that gfh1Δ and mod21Δ mutants have qualitatively normal microtubule nucleation from all types of microtubule-organizing centers (MTOCs) in vivo but quantitatively reduced nucleation from interphase MTOCs, and this is exacerbated by mutations in mod22+. Simultaneous deletion of gfh1p, mod21p, and alp16p, a third nonessential γ-TuC protein, does not lead to additive defects, suggesting that all three proteins contribute to a single function. Coimmunoprecipitation experiments suggest that gfh1p and alp16p are codependent for association with a small “core” γ-TuC, whereas mod21p is more peripherally associated, and that gfh1p and mod21p may form a subcomplex independently of the small γ-TuC. Interestingly, sucrose gradient analysis suggests that the major form of the γ-TuC in fission yeast may be a small complex. We propose that gfh1p, mod21p, and alp16 act as facultative “noncore” components of the fission yeast γ-TuC and enhance its microtubule-nucleating ability.
From an insertional mutagenesis screen, we isolated a novel gene, mto2+, involved in microtubule organization in fission yeast. mto2Δ strains are viable but exhibit defects in interphase microtubule nucleation and in formation of the postanaphase microtubule array at the end of mitosis. The mto2Δ defects represent a subset of the defects displayed by cells deleted for mto1+ (also known as mod20+ and mbo1+), a centrosomin-related protein required to recruit the γ-tubulin complex to cytoplasmic microtubule-organizing centers (MTOCs). We show that mto2p colocalizes with mto1p at MTOCs throughout the cell cycle and that mto1p and mto2p coimmunoprecipitate from cytoplasmic extracts. In vitro studies suggest that mto2p binds directly to mto1p. In mto2Δ mutants, although some aspects of mto1p localization are perturbed, mto1p can still localize to spindle pole bodies and the cell division site and to “satellite” particles on interphase microtubules. In mto1Δ mutants, localization of mto2p to all of these MTOCs is strongly reduced or absent. We also find that in mto2Δ mutants, cytoplasmic forms of the γ-tubulin complex are mislocalized, and the γ-tubulin complex no longer coimmunoprecipitates with mto1p from cell extracts. These experiments establish mto2p as a major regulator of mto1p-mediated microtubule nucleation by the γ-tubulin complex.
Although endocytosis and exocytosis have been extensively studied in budding yeast, there have been relatively few investigations of these complex processes in the fission yeast Schizosaccharomyces pombe. Here we identify and characterize fission yeast Mug33, a novel Tea1-interacting protein, and show that Mug33 is involved in exocytosis. Mug33 is a Sur7/PalI-family transmembrane protein that localizes to the plasma membrane at the cell tips and to cytoplasmic tubulovesicular elements (TVEs). A subset of Mug33 TVEs make long-range movements along actin cables, co-translocating with subunits of the exocyst complex. TVE movement depends on the type V myosin Myo52. Although mug33Δ mutants are viable, with only a mild cell-polarity phenotype, mug33Δ myo52Δ double mutants are synthetically lethal. Combining mug33 Δ with deletion of the formin For3 (for3Δ) leads to synthetic temperature-sensitive growth and strongly reduced levels of exocytosis. Interestingly, mutants in non-essential genes involved in exocyst function behave in a manner similar to mug33Δ when combined with myo52Δ and for3Δ. By contrast, combining mug33Δ with mutants in non-essential exocyst genes has only minor effects on growth. We propose that Mug33 contributes to exocyst function and that actin cable-dependent vesicle transport and exocyst function have complementary roles in promoting efficient exocytosis in fission yeast.
Mug33; Actin filament; Schizosaccharomyces pombe; Sur7/PalI; Exocytosis; Fission yeast
Although the fission yeast Schizosaccharomyces pombe contains many of the γ-tubulin ring complex (γ-TuRC)-specific proteins of the γ-tubulin complex (γ-TuC), several questions about the organizational state and function of the fission yeast γ-TuC in vivo remain unresolved. Using 3×GFP-tagged γ-TuRC-specific proteins, we show here that γ-TuRC-specific proteins are present at all microtubule organizing centers in fission yeast and that association of γ-TuRC-specific proteins with the γ-tubulin small complex (γ-TuSC) does not depend on Mto1, which is a key regulator of the γ-TuC. Through sensitive imaging in mto1Δ mutants, in which cytoplasmic microtubule nucleation is abolished, we unexpectedly found that γ-TuC incapable of nucleating microtubules can nevertheless associate with microtubule minus-ends in vivo. The presence of γ-TuC at microtubule ends is independent of γ-TuRC-specific proteins and strongly correlates with the stability of microtubule ends. Strikingly, microtubule bundles lacking γ-TuC at microtubule ends undergo extensive treadmilling in vivo, apparently induced by geometrical constraints on plus-end growth. Our results indicate that microtubule stabilization by the γ-TuC, independently of its nucleation function, is important for maintaining the organization and dynamic behavior of microtubule arrays in vivo.
Fission yeast; Gamma-tubulin; Microtubules
Microtubule nucleation by the γ-tubulin complex occurs primarily at centrosomes, but more diverse types of microtubule organizing centers (MTOCs) also exist, especially in differentiated cells [1–4]. Mechanisms generating MTOC diversity are poorly understood. Fission yeast Schizosaccharomyces pombe has multiple types of cytoplasmic MTOCs, and these vary through the cell cycle [5, 6]. Cytoplasmic microtubule nucleation in fission yeast depends on a complex of proteins Mto1 and Mto2 (Mto1/2), which localizes to MTOCs and interacts with the γ-tubulin complex [7–12]. Localization of Mto1 to prospective MTOC sites has been proposed as a key step in γ-tubulin complex recruitment and MTOC formation [9, 13], but how Mto1 localizes to such sites has not been investigated. Here we identify a short conserved C-terminal sequence in Mto1, termed MASC, important for targeting Mto1 to multiple distinct MTOCs. Different subregions of MASC target Mto1 to different MTOCs, and multimerization of MASC is important for efficient targeting. Mto1 targeting to the cell equator during division depends on direct interaction with unconventional type II myosin Myp2. Targeting to the spindle pole body during mitosis depends on Sid4 and Cdc11, components of the septation initiation network (SIN), but not on other SIN components.
► Conserved MASC motif in Mto1 contains multiple independent MTOC-targeting sequences ► Multimerization is important for MASC-dependent Mto1 localization ► Interaction with Myp2 controls Mto1 localization to cell equator in cell division ► Mto1 at mitotic spindle pole bodies requires Sid4 and Cdc11, but not other SIN proteins
Fission yeast genes identified in genetic screens are usually cloned by transformation of mutants with plasmid libraries. However, for some genes this can be difficult, and positional cloning approaches are required. The mutation swi5-39 reduces recombination frequency in homozygous crosses and has been used as a tool in mapping gene position (Schmidt, 1993). However, strain construction in swi5-39-based mapping is significantly more laborious than is desirable. Here we describe a set of strains designed to make swi5-based mapping more efficient and more powerful. The first improvement is the use of a swi5Δ strain marked with kanamycin (G418) resistance, which greatly facilitates identification of swi5 mutants. The second improvement, which follows directly from the first, is the introduction of a large number of auxotrophic markers into mapping strains, increasing the likelihood of finding close linkage between a marker and the mutation of interest. We combine these new mapping strains with a rec12Δ-based approach for initial mapping of a mutation to an individual chromosome. Together, the two methods allow an approximate determination of map position in only a small number of crosses. We used these to determine that mod22-1, a modifier of microtubule nucleation phenotypes, encodes a truncation allele of Swr1, a chromatin-remodelling factor involved in nucleosomal deposition of H2A.Z histone variant Pht1. Expression microarray analysis of mod22-1, swr1Δ and pht1Δ cells suggests that the modifier phenotype of mod22-1 mutants may be due to small changes in expression of one or more genes involved in tubulin function. Copyright © 2009 John Wiley & Sons, Ltd.
Schizosaccharomyces pombe; gene mapping; γ-tubulin; Swr1; H2A.Z
Many systems regulating cell polarity involve stable landmarks defined by internal cues [1–5]. In the rod-shaped fission yeast Schizosaccharomyces pombe, microtubules regulate polarized vegetative growth via a landmark involving the protein Tea1 [6–9]. Tea1 is delivered to cell tips as packets of molecules associated with growing microtubule ends  and anchored at the plasma membrane via a mechanism involving interaction with the membrane protein Mod5 [11, 12]. Tea1 and Mod5 are highly concentrated in clusters at cell tips in a mutually dependent manner, but how the Tea1-Mod5 interaction contributes mechanistically to generating a stable landmark is not understood. Here, we use live-cell imaging, FRAP, and computational modeling to dissect dynamics of the Tea1-Mod5 interaction. Surprisingly, we find that Tea1 and Mod5 exhibit distinctly different turnover rates at cell tips. Our data and modeling suggest that rather than acting simply as a Tea1 receptor or as a molecular “glue” to retain Tea1, Mod5 functions catalytically to stimulate incorporation of Tea1 into a stable tip-associated cluster network. The model also suggests an emergent self-focusing property of the Tea1-Mod5 cluster network, which can increase the fidelity of polarized growth.
► Interacting polarity proteins Tea1 and Mod5 have different mobilities at cell tips ► Modeling suggests Tea1 assembles into polymeric cluster networks ► Mod5 acts catalytically in formation of Tea1 cluster networks ► Self-focusing property of cluster networks increases fidelity of polarized growth
Stable isotope labelling by amino acids in cell culture (SILAC) provides a straightforward tool for quantitation in proteomics. However, one problem associated with SILAC is the in vivo conversion of labelled arginine to other amino acids, typically proline. Here we show that arginine-conversion in the fission yeast Schizosaccharomyces pombe occurs at extremely high levels, such that labelling cells with heavy arginine leads to undesired incorporation of label into essentially all of the proline pool, as well as a substantial portion of glutamate, glutamine and lysine pools. We find that this can be prevented by deleting genes involved in arginine catabolism, using methods that are highly robust yet simple to implement. Deletion of both fission yeast arginase genes or of the single ornithine transaminase gene, together with a small modification to to growth medium that improves arginine uptake in mutant strains, is sufficient to abolish essentially all arginine-conversion. We demonstrate the usefulness of our approach in a large-scale quantitative analysis of proteins before and after cell division, which successfully identified both up- and down-regulated proteins, including a novel protein involved in septation. This strategy for addressing the “arginine-conversion problem” may be more broadly applicable to organisms amenable to genetic manipulation.
Stable isotope labeling by amino acids in cell culture (SILAC) provides a straightforward tool for quantitation in proteomics. However, one problem associated with SILAC is the in vivo conversion of labeled arginine to other amino acids, typically proline. We found that arginine conversion in the fission yeast Schizosaccharomyces pombe occurred at extremely high levels, such that labeling cells with heavy arginine led to undesired incorporation of label into essentially all of the proline pool as well as a substantial portion of glutamate, glutamine, and lysine pools. We found that this can be prevented by deleting genes involved in arginine catabolism using methods that are highly robust yet simple to implement. Deletion of both fission yeast arginase genes or of the single ornithine transaminase gene, together with a small modification to growth medium that improves arginine uptake in mutant strains, was sufficient to abolish essentially all arginine conversion. We demonstrated the usefulness of our approach in a large scale quantitative analysis of proteins before and after cell division; both up- and down-regulated proteins, including a novel protein involved in septation, were successfully identified. This strategy for addressing the “arginine conversion problem” may be more broadly applicable to organisms amenable to genetic manipulation.
Immunoglobulin G (IgG)–Sepharose is often used for purification of protein A- and tandem affinity purification (TAP)-tagged proteins from eukaryotic cells, but because it is based on an agarose matrix, it is not always optimal for all proteins. Synthetic matrices such as IgG–Dynabeads have improved properties over IgG–Sepharose but are generally expensive. Here we describe the preparation and properties of an IgG matrix based on Fractogel EMD beads. As a synthetic-based matrix, IgG–Fractogel has clear advantages over IgG–Sepharose. IgG–Fractogel can also be used in applications that usually use IgG–Dynabeads but at a significantly reduced cost.
Cytoplasmic microtubule nucleation in the fission yeast Schizosaccharomyces pombe involves the interacting proteins Mto1 and Mto2, which are thought to recruit the γ-tubulin complex (γ-TuC) to prospective microtubule organizing centers. Mto1 contains a short amino-terminal region (CM1) that is conserved in higher eukaryotic proteins implicated in microtubule organization, centrosome function and/or brain development. Here we show that mutations in the Mto1 CM1 region generate mutant proteins that are functionally null for cytoplasmic microtubule nucleation and interaction with the γ-TuC (phenocopying mto1Δ), even though the Mto1-mutant proteins localize normally in cells and can bind Mto2. Interestingly, the CM1 region is not sufficient for efficient interaction with the γ-TuC. Mutation within a different region of Mto1, outside CM1, abrogates Mto2 binding and also impairs cytoplasmic microtubule nucleation and Mto1 association with the γ-TuC. However, this mutation allows limited microtubule nucleation in vivo, phenocopying mto2Δ rather than mto1Δ. Further experiments suggest that Mto1 and Mto2 form a complex (Mto1/2 complex) independent of the γ-TuC and that Mto1 and Mto2 can each associate with the γ-TuC in the absence of the other, albeit extremely weakly compared to when both Mto1 and Mto2 are present. We propose that Mto2 acts cooperatively with Mto1 to promote association of Mto1/2 complex with the γ-TuC.
fission yeast; γ-tubulin complex; centrosomin-related proteins
Müller et al. (Reports, 27 October 2006, p. 654) showed that inhibition of the γ-tubulin ring complex (γ-TuRC) activates the spindle assembly checkpoint (SAC), which led them to suggest that γ-TuRC proteins play molecular roles in SAC activation. Because γ-TuRC inhibition leads to pleiotropic spindle defects, which are well known to activate kinetochore-derived checkpoint signaling, we believe that this conclusion is premature.
To investigate the role of microtubules in regulating cell polarity in Schizosaccharomyces pombe, we have developed a system in which normally cylindrical fission yeast synchronously form branched cells at high frequency upon treatment with the microtubule-depolymerizing drug thiabendazole (TBZ). Branching depends on both elevated temperature and cell cycle state and occurs at high frequency only when TBZ is added to cells that have not yet passed through New-End Take-Off (NETO), the normal transition from monopolar to bipolar growth. This suggests that microtubules may be of greatest physiological importance for the maintenance of cell shape at specific points in the cell cycle. The localization of three different proteins normally found at cell ends—cortical F-actin, tea1, and an ral3 (scd2)–green fluorescent protein (GFP) fusion—is disrupted by TBZ treatment. However, these proteins can eventually return to cell ends in the absence of microtubules, indicating that although their localization to ends normally depends on microtubules, they may recover by alternative mechanisms. In addition, TBZ induces a shift in ral3–GFP distribution from cell ends to the cell middle, suggesting that a protein complex containing ral3 may be part of the cue that specifies the position of branch formation.
S. pombe; microtubules; thiabendazole; actin; cell polarity