Yukiko Nakase

Live Observation of Forespore Membrane Formation in Fission Yeast

Sporulation in the fission yeast Schizosaccharomyces pombe is a unique biological process in that the plasma membrane of daughter cells is assembled de novo within the mother cell cytoplasm. A double unit membrane called the forespore membrane (FSM) is constructed dynamically during meiosis. To obtain a dynamic view of FSM formation, we visualized FSM in living cells by using green fluorescent protein fused with Psy1, an FSM-resident protein, together with the nucleus or microtubules. The assembly of FSM initiates in prophase II, and four FSMs in a cell expand in a synchronous manner at the same rate throughout meiosis II. After the meiosis II completes, FSMs continue to expand until closure to form the prespore, a spore precursor. Prespores are initially ellipsoidal, and eventually become spheres. FSM formation was also observed in the sporulation-deficient mutants spo3, spo14, and spo15. In the spo15 mutant, the initiation of FSM formation was completely blocked. In the spo3 mutant, the FSM expanded normally during early meiosis II, but it was severely inhibited during late and postmeiosis, whereas in the spo14 mutant, membrane expansion was more severely inhibited throughout meiosis II. These observations suggest that FSM expansion is composed of two steps, early meiotic FSM expansion and late and post meiotic FSM expansion. Possible regulatory mechanisms of FSM formation in fission yeast are discussed.

Meiotic Spindle Pole Bodies Acquire the Ability to Assemble the Spore Plasma Membrane by Sequential Recruitment of Sporulation-specific Components in Fission Yeast

The spindle pole body (SPB) of Schizosaccharomyces pombe is required for assembly of the forespore membrane (FSM) during meiosis. Before de novo biogenesis of the FSM, the meiotic SPB forms outer plaques, an event referred to as SPB modification. A constitutive SPB component, Spo15, plays an indispensable role in SPB modification and sporulation. Here, we analyzed two sporulation-specific genes, spo13(+) and spo2(+), which are not required for progression of meiotic nuclear divisions, but are essential for sporulation. Spo13 is a 16-kDa coiled-coil protein, and Spo2 is a 15-kDa nonconserved protein. Both Spo13 and Spo2 specifically associated with the meiotic SPB. The respective deletion mutants are viable, but defective in SPB modification and in the onset of FSM formation. Spo13 and Spo2 localized on the cytoplasmic side of the SPB in close contact with the nascent FSM. Localization of Spo13 to the SPB was dependent on Spo15 and Spo2; that of Spo2 depended only on Spo15, suggesting that their recruitment to the SPB is strictly controlled. Spo2 physically associated with both Spo15 and Spo13, but Spo13 and Spo15 did not interact directly. Taken together, these observations indicate that Spo2 is recruited to the SPB during meiosis and then assists in the localization of Spo13 to the outer surface of the SPB.

A defect in protein farnesylation suppresses a loss of Schizosaccharomyces pombe tsc2+, a homolog of the human gene predisposing to tuberous sclerosis complex.

Mutations in the human Tsc1 and Tsc2 genes predispose to tuberous sclerosis complex (TSC), a disorder characterized by the wide spread of benign tumors. Tsc1 and Tsc2 proteins form a complex and serve as a GTPase-activating protein (GAP) for Rheb, a GTPase regulating a downstream kinase, mTOR. The genome of Schizosaccharomyces pombe contains tsc1+ and tsc2+, homologs of human Tsc1 and Tsc2, respectively. In this study we analyzed the gene expression profile on a genomewide scale and found that deletion of either tsc1+ or tsc2+ affects gene induction upon nitrogen starvation. Three hours after nitrogen depletion genes encoding permeases and genes required for meiosis are less induced. Under the same condition, retrotransposons, G1-cyclin (pas1+), and inv1+ are more induced. We also demonstrate that a mutation (cpp1-1) in a gene encoding a β-subunit of a farnesyltransferase can suppress most of the phenotypes associated with deletion of tsc1+ or tsc2+. When a mutant of rhb1+ (homolog of human Rheb), which bypasses the requirement of protein farnesylation, was expressed, the cpp1-1 mutation could no longer suppress, indicating that deficient farnesylation of Rhb1 contributes to the suppression. On the basis of these results, we discuss TSC pathology and possible improvement in chemotherapy for TSC.

Distinctive Responses to Nitrogen Starvation in the Dominant Active Mutants of the Fission Yeast Rheb GTPase

Rheb, a Ras-like small GTPase conserved from human to yeast, controls Tor kinase and plays a central role in the regulation of cell growth depending on extracellular conditions. Rhb1 (a fission yeast homolog of Rheb) regulates amino acid uptake as well as response to nitrogen starvation. In this study, we generated two mutants, rhb1-DA4 and rhb1-DA8, and characterized them genetically. The V17A mutation within the G1 box defined for the Ras-like GTPases was responsible for rhb1-DA4 and Q52R I76F within the switch II domain for rhb1-DA8. In fission yeast, two events—the induction of the meiosis-initiating gene mei2+ and cell division without cell growth—are a typical response to nitrogen starvation. Under nitrogen-rich conditions, Rheb stimulates Tor kinase, which, in turn, suppresses the response to nitrogen starvation. While amino acid uptake was prevented by both rhb1-DA4 and rhb1-DA8 in a dominant fashion, the response to nitrogen starvation was prevented only by rhb1-DA4. rhb1-DA8 thereby allowed genetic dissection of the Rheb-dependent signaling cascade. We postulate that the signaling cascade may branch below Rhb1 or Tor2 and regulate the amino acid uptake and response to nitrogen starvation independently.

Intracellular trafficking and ubiquitination of the Schizosaccharomyces pombe amino acid permease Aat1p

In Schizosaccharomyces pombe, neither intracellular sorting nor ubiquitination of amino acid permeases is well understood. In the present study, we show that intracellular sorting of the amino acid permease Aat1p in S. pombe depends on the presence of a nitrogen source in the growth medium. Under nitrogen-sufficient conditions, Aat1p appeared to be stably localized at the Golgi apparatus. In contrast, under nitrogen-insufficient conditions, Aat1p was sorted to the plasma membrane. Over time, plasma membrane-localized Aat1p was internalized and sorted to the lumen of the vacuole, where it was degraded. Sorting of Aat1p to the vacuolar lumen was dependent on the ESCRT (endosomal sorting complex required for transport) complex, which is required for formation of the multivesicular body. S. pombe has three genes (pub1(+), pub2(+) and pub3(+)) that are homologous to the ubiquitin ligase RSP5. Under nitrogen-sufficient conditions, Aat1-GFP was missorted to the plasma membrane in pub1Δ cells and ubiquitinated Aat1p was not detected. These results suggest that Pub1p-mediated ubiquitination is required for retention of Aat1 at the Golgi under nitrogen-sufficient conditions. The Aat1p lysine mutant Aat1(K18, 26, 27) was completely missorted to the plasma membrane under nitrogen-rich conditions. Furthermore, Aat1(K4, 18R), Aat1(K4, 26, 27R) and Aat1(K18, 26, 27K) mutants were severely blocked in endocytosis. These results indicate that ubiquitination is an important determinant for localization and regulation of the Aat1p permease in S. pombe.

The fission yeast β-arrestin-like protein Any1 is involved in TSC-Rheb signaling and the regulation of amino acid transporters

Summary Rheb GTPase and the Tsc1–Tsc2 protein complex, which serves as a GTPase-activating protein for Rheb, have crucial roles in the regulation of cell growth in response to extracellular conditions. In Schizosaccharomyces pombe, Rheb and Tsc1–Tsc2 regulate cell cycle progression, the onset of meiosis and the uptake of amino acids. In cells lacking Tsc2 (&Dgr;tsc2), the amino acid transporter Aat1, which is normally expressed on the plasma membrane under starvation conditions, is confined to the Golgi. Here, we show that the loss of either pub1+, encoding an E3 ubiquitin ligase, or any1+, encoding a &bgr;-arrestin-like protein, allows constitutive expression of Aat1 on the plasma membrane in &Dgr;tsc2 cells, suggesting that Pub1 and Any1 are required for localization of Aat1 to the Golgi. Subsequent analysis revealed that, in the Golgi, Pub1 and Any1 form a complex that ubiquitylates Aat1. Physical interaction of Pub1 and Any1 is more stable in &Dgr;tsc2 cells than in wild-type cells and is independent of Tor2 activity. These results indicate that the TSC-Rheb signaling pathway regulates the localization of amino acid transporters via Pub1 and Any1 in a Tor2-independent manner. Our study demonstrates that, unlike in budding yeast (in which Rsp5 and ARTs, a pair of proteins analogous to Pub1 and Any1, respectively, primarily act to reduce expression of the transporters on plasma membrane when nutrients are abundant), the primary role of fission yeast Pub1 and Any1 is to store the transporter in the Golgi under nutrient-rich conditions.

Ectopic Overproduction of a Sporulation-specific Transcription Factor Induces Assembly of Prespore-like Membranous Compartments in Vegetative Cells of Fission Yeast

Mei4 is a key sporulation-specific transcription factor in fission yeast. Ectopic expression of Mei4 in vegetative cells caused formation of nucleated membranous compartments, which shared common features with normal forespore membranes, thereby perturbing nuclear division. These results suggest why expression of development-specific transcription factors must be strictly controlled.

RHEB-mTOR axis regulates expression of transposons Tf2s in fission yeast.

ABSTRACT The human TSC2 gene, mutations in which predispose individuals to the disease tuberous sclerosis complex (TSC), encodes a GTPase-activating protein for the GTPase RHEB. Loss of TSC2 results in constitutive activation of RHEB and its target mammalian target of rapamycin (mTOR). We have previously reported that fission yeast (Schizosaccharomyces pombe) Tf2 retrotransposons (hereafter Tf2s) are abnormally induced upon nitrogen starvation in cells lacking the tsc2+ gene (Δtsc2), a homolog of the human TSC2 gene, and in cells with a dominant-active mutation in the fission yeast RHEB GTPase (rhb1-DA4). We report here that induction of Tf2s in these mutants is suppressed upon overexpression of the cgs2+ gene, which encodes a cAMP-specific phosphodiesterase, or upon deletion of components in the glucose/cAMP signaling pathway, namely Cyr1, Pka1, Tor1 and the stress-activated transcription factor Atf1. The results suggest that the glucose/cAMP signaling pathway is downregulated when cells are starved for nitrogen. We also show that Tf2 proteins are degraded via autophagy, which is under control of Tor2, a homolog of human mTOR. It appears that failure in the two processes, downregulation of the glucose/cAMP signaling pathway and induction of autophagy, allows abnormal induction of Tf2s upon nitrogen starvation in Δtsc2 and rhb1-DA4 cells. Summary: Abnormal induction of the Tf2 retrotransposons upon nitrogen starvation in cells lacking the tsc2+ gene is due to defects in RHEB–mTOR and the glucose/cAMP signaling and induction of autophagy.

Fission Yeast CENP-C (Cnp3) Plays a Role in Restricting the Site of CENP-A Accumulation

The centromere is a chromosomal locus where a microtubule attachment site, termed kinetochore, is assembled in mitosis. In most eukaryotes, with the exception of holocentric species, each chromosome contains a single distinct centromere. A chromosome with an additional centromere undergoes successive rounds of anaphase bridge formation and breakage, or triggers a cell cycle arrest imposed by DNA damage and replication checkpoints. We report here a study in Schizosaccharomyces pombe to characterize a mutant (cnp3-1) in a gene encoding a homolog of mammalian centromere-specific protein, CENP-C. At the restrictive temperature 36°, the Cnp3-1 mutant protein loses its localization at the centromere. In the cnp3-1 mutant, the level of the Cnp1 (a homolog of a centromere-specific histone CENP-A) also decreases at the centromere. Interestingly, the cnp3-1 mutant is prone to promiscuous accumulation of Cnp1 at non-centromeric regions, when Cnp1 is present in excess. Unlike the wild type protein, Cnp3-1 mutant protein is found at the sites of promiscuous accumulation of Cnp1, suggesting that Cnp3-1 may stabilize or promote accumulation of Cnp1 at non-centromeric regions. From these results, we infer the role of Cnp3 in restricting the site of accumulation of Cnp1 and thus to prevent formation of de novo centromeres.