The Schizosaccharomyces pombe spo20-KC104 mutation was originally isolated in a screen for sporulation-deficient mutants, and the spo20-KC104 mutant exhibits temperature-sensitive growth. Herein, we report that S. pombe, spo20(+) is essential for fission yeast cell viability and is constitutively expressed throughout the life cycle. We also demonstrate that the spo20(+) gene product is structurally homologous to Saccharomyces cerevisiae Sec14, the major phosphatidylinositol transfer protein of budding yeast. This structural homology translates to a significant degree of functional relatedness because reciprocal complementation experiments demonstrate that each protein is able to fulfill the essential function of the other. Moreover, biochemical experiments show that, like Sec14, Spo20 is a phosphatidylinositol/phosphatidylcholine-transfer protein. That Spo20 is required for Golgi secretory function in vegetative cells is indicated by our demonstration that the spo20-KC104 mutant accumulates aberrant Golgi cisternae at restrictive temperatures. However, a second phenotype observed in Spo20-deficient fission yeast is arrest of cell division before completion of cell separation. Consistent with a direct role for Spo20 in controlling cell septation in vegetatively growing cells, localization experiments reveal that Spo20 preferentially localizes to the cell poles and to sites of septation of fission yeast cells. We also report that, when fission yeasts are challenged with nitrogen starvation, Spo20 translocates to the nucleus. This nuclear localization persists during conjugation and meiosis. On completion of meiosis, Spo20 translocates to forespore membranes, and it is the assembly of forespore membranes that is abnormal in spo20-KC104 cells. In such mutants, a considerable fraction of forming prespores fail to encapsulate the haploid nucleus. Our results indicate that Spo20 regulates the formation of specialized membrane structures in addition to its recognized role in regulating Golgi secretory function.

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.

SummaryRheb 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 (Dtsc2), 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 b-arrestin-like protein, allows constitutive expression of Aat1 on the plasma membrane in Dtsc2 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 Dtsc2 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.

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.

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 1 and tsc2 1