SummaryIn Saccharomyces cerevisiae, three classes of sphingolipids contain myo-inositol -inositol phosphorylceramide (IPC), mannosylinositol phosphorylceramide (MIPC) and mannosyldiinositol phosphorylceramide [M(IP) 2 C]. No fission yeast equivalent of Ipt1p, the inositolphosphotransferase that synthesizes M(IP) 2 C from MIPC, has been found in the Schizosaccharomyces pombe genome. Analysis of the sphingolipid composition of wild-type cells confirmed that MIPC is the terminal and most abundant complex sphingolipid in S. pombe. Three proteins (Sur1p, Csg2p and Csh1p) have been shown to be involved in the synthesis of MIPC from IPC in S. cerevisiae. The S. pombe genome has three genes (SPAC2F3.01, SPCC4F11.04c and SPAC17G8.11c) that are homologues of SUR1, termed imt1 + , imt2 + and imt3 + , respectively. To determine whether these genes function in MIPC synthesis in S. pombe, single and multiple gene disruptants were constructed. Single imt disruptants were found to be viable. MIPC was not detected and IPC levels were increased in the triple disruptant, indicating that the three SUR1 homologues are involved in the synthesis of MIPC. GFP-tagged Imt1p, Imt2p and Imt3p localized to Golgi apparatus membranes. The MIPC-deficient mutant exhibited pleiotropic phenotypes, including defects in cellular and vacuolar morphology, and in localization of ergosterols. MIPC seemed to be required for endocytosis of a plasmamembrane-localized amino acid transporter, because sorting of the transporter from the plasma membrane to the vacuole was severely impaired in the MIPC-deficient mutant grown under nitrogen-limiting conditions. These results suggest that MIPC has multiple functions not only in the maintenance of cell and vacuole morphology but also in vesicular trafficking in fission yeast.

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.

a b s t r a c tAutophagy is a non-selective degradation process in eukaryotic cells. The genome sequence of the fission yeast Schizosaccharomyces pombe has revealed that many of the genes required for autophagy are common between the fission yeast and budding yeast, suggesting that the basic machinery of autophagy is conserved between these species. Autophagy in fission yeast is specifically induced by nitrogen starvation based on monitoring a GFP-Atg8p marker. Upon nitrogen starvation, fission yeast cells exit the vegetative cell cycle and initiate sexual differentiation to produce spores. Most of the nitrogen used for de novo protein synthesis during sporulation derives from the autophagic protein degradation system. This review focuses on the recent advances in the role of autophagy in fission yeast.

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 pub1D 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.

c Uptake of extracellular oligopeptides in yeast is mediated mainly by specific transporters of the peptide transporter (PTR) and oligopeptide transporter (OPT) families. Here, we investigated the role of potential peptide transporters in the yeast Schizosaccharomyces pombe. Utilization of naturally occurring dipeptides required only Ptr2/SPBC13A2.04c and none of the other 3 OPT proteins (Isp4, Pgt1, and Opt3), whereas only Isp4 was indispensable for tetrapeptide utilization. Both Ptr2 and Isp4 localized to the cell surface, but under rich nutrient conditions Isp4 localized in the Golgi apparatus through the function of the ubiquitin ligase Pub1. Furthermore, the ubiquitin ligase Ubr11 played a significant role in oligopeptide utilization. The mRNA levels of both the ptr2 and isp4 genes were significantly reduced in ubr11⌬ cells, and the dipeptide utilization defect in the ubr11⌬ mutant was rescued by the forced expression of Ptr2. Consistent with its role in transcriptional regulation of peptide transporter genes, the Ubr11 protein was accumulated in the nucleus. Unlike the situation in Saccharomyces cerevisiae, the oligopeptide utilization defect in the S. pombe ubr11⌬ mutant was not rescued by inactivation of the Tup11/12 transcriptional corepressors, suggesting that the requirement for the Ubr ubiquitin ligase in the upregulation of peptide transporter mRNA levels is conserved in both yeasts; however, the actual mechanism underlying the control appears to be different. We also found that the peptidomimetic proteasome inhibitor MG132 was still operative in a strain lacking all known PTR and OPT peptide transporters. Therefore, irrespective of its peptide-like structure, MG132 is carried into cells independently of the representative peptide transporters.