Changes in S1P(1) and SIP(2) expression during embryonal development and primitive endoderm differentiation of F9 cells

Sphingosine 1-phosphate (S1P) is a ligand for S1P family receptors (S1P1–S1P5). Of these receptors, S1P1, S1P2, and S1P3 are ubiquitously expressed in adult mice, while S1P4 and S1P5 are tissue specific. However, little is known of their expression during embryonal development. We performed Northern blot analyses in mouse embryonal tissue and found that such expression is developmentally regulated. We also examined the expression of these receptors during primitive endoderm (PrE) differentiation of mouse F9 embryonal carcinoma (EC) cells, a well-known in vitro endoderm differentiation system. S1P2 mRNA was abundantly expressed in F9 EC cells, but little S1P1 and no S1P3, S1P4, or S1P5 mRNA was detectable. However, S1P1 mRNA expression was induced during EC-to-PrE differentiation. Studies using small interference RNA of S1P1 indicated that increased S1P1 expression is required for PrE differentiation. Thus, S1P1 may play an important function in PrE differentiation that is not substituted for by S1P2

Intracellular localization and tissue-specific distribution of human and yeast DHHC cysteine-rich domain-containing proteins

Increasing evidence indicates that DHHC cysteine-rich domain-containing proteins (DHHC proteins) are protein acyltransferases. Although multiple DHHC proteins are found in eukaryotes, characterization has been examined for only a few. Here, we have cloned all the yeast and human DHHC genes and investigated their intracellular localization and tissue-specific expression. Most DHHC proteins are localized in the ER and/or Golgi, with a few localized in the plasma membrane and one in the yeast vacuole. Human DHHC mRNAs also differ in their tissue-specific expression. These results may provide clues to aid in discovering the specific function(s) of each DHHC protein

Intracellular Trafficking Pathway of Yeast Long-chain Base Kinase Lcb4, from Its Synthesis to Its Degradation

Sphingoid long-chain base 1-phosphates act as bioactive lipid molecules in eukaryotic cells. In budding yeast, long-chain base 1-phosphates are synthesized mainly by the long-chain base kinase Lcb4. We recently reported that, soon after yeast cells enter into the stationary phase, Lcb4 is rapidly degraded by being delivered to the vacuole in a palmitoylation- and phosphorylation-dependent manner. In this study, we investigated the complete trafficking pathway of Lcb4, from its synthesis to its degradation. After membrane anchoring by palmitoylation at the Golgi apparatus, Lcb4 is delivered to the plasma membrane (PM) through the late Sec pathway and then to the endoplasmic reticulum (ER). The yeast ER consists of a cortical network juxtaposed to the PM (cortical ER) with tubular connections to the nuclear envelope (nuclear ER). Remarkably, the localization of Lcb4 is restricted to the cortical ER. As the cells reach the stationary phase, G1 cell cycle arrest initiates Lcb4 degradation and its delivery to the vacuole via the Golgi apparatus. The protein transport pathway from the PM to the ER found in this study has not been previously reported. We speculate that this novel pathway is mediated by the PM-ER contact

Lack of sphingosine 1-phosphate-degrading enzymes in erythrocytes

Platelets are known to store a large amount of the bioactive lipid molecule sphingosine 1-phosphate (S1P) and to release it into the plasma in a stimuli-dependent manner. Erythrocytes can also release S1P, independently from any stimuli. We measured the S1P and sphingosine (Sph) levels in erythrocytes by HPLC and found that the contribution of erythrocyte S1P to whole blood S1P levels is actually higher than that of platelets. In vitro assays demonstrated that erythrocytes possess much weaker Sph kinase activity compared to platelets but lack the S1P-degrading activities of either S1P lyase or S1P phosphohydrolase. This combination may enable erythrocytes to maintain a high S1P content relative to Sph. The absence of both S1P-degrading enzymes has not been reported for other cell types. Thus, erythrocytes may be specialized cells for storing and supplying plasma S1P

Inositol Depletion Restores Vesicle Transport in Yeast Phospholipid Flippase Mutants

In eukaryotic cells, type 4 P-type ATPases function as phospholipid flippases, which translocate phospholipids from the exoplasmic leaflet to the cytoplasmic leaflet of the lipid bilayer. Flippases function in the formation of transport vesicles, but the mechanism remains unknown. Here, we isolate an arrestin-related trafficking adaptor, ART5, as a multicopy suppressor of the growth and endocytic recycling defects of flippase mutants in budding yeast. Consistent with a previous report that Art5p downregulates the inositol transporter Itr1p by endocytosis, we found that flippase mutations were also suppressed by the disruption of ITR1, as well as by depletion of inositol from the culture medium. Interestingly, inositol depletion suppressed the defects in all five flippase mutants. Inositol depletion also partially restored the formation of secretory vesicles in a flippase mutant. Inositol depletion caused changes in lipid composition, including a decrease in phosphatidylinositol and an increase in phosphatidylserine. A reduction in phosphatidylinositol levels caused by partially depleting the phosphatidylinositol synthase Pis1p also suppressed a flippase mutation. These results suggest that inositol depletion changes the lipid composition of the endosomal/TGN membranes, which results in vesicle formation from these membranes in the absence of flippases

Effect of Oxygen Concentration in NH3-SCR Reaction over Fe- and Cu-loaded Beta Zeolites

The kinetic study on the effect of O-2 concentration in the selective catalytic reduction (SCR) of nitrogen oxide (NOx) by ammonia (NH3) was systematically carried out at temperatures below 200 degrees C over Fe- and Cu-loaded beta zeolites. The large difference was observed in the effect of O-2 concentration over the two catalysts: for the Fe-loaded beta zeolite, the NO conversion increased considerably with the O-2 concentration. On the other hand, for the Cu-loaded beta zeolite, increasing the concentration of O-2 did not have a significant impact. In addition, the temperature dependence of the apparent reaction orders was investigated. The apparent reaction order of O-2 decreased with an increase in the reaction temperature, being 0.9 at 150 degrees C and 0.4 at 200 degrees C for the Fe-loaded beta zeolite, and 0.2 at 125 degrees C and 0.1 at 175 degrees C for the Cu-loaded beta zeolite. The fact that the degree of the reduction in the reaction order of O-2 was consisted with that of the increase in the reaction order of NH3 when the reaction temperature was increased strongly suggests that adsorbed NH3 inhibits the adsorption of O-2

Long-Chain Base Kinase Lcb4 Is Anchored to the Membrane through Its Palmitoylation by Akr1

Sphingoid long-chain base kinase Lcb4 catalyzes the production of the bioactive lipid molecules the long-chain base 1-phosphates. Although Lcb4 has no apparent transmembrane-spanning domain, it is tightly associated with the membrane. Here, we demonstrate that Lcb4 is modified by palmitoylation. This modification was greatly reduced in mutants for AKR1, which was recently identified as encoding a protein acyltransferase. In vitro experiments revealed that Akr1 indeed acts as a protein acyltransferase for Lcb4. Studies using site-directed mutagenesis indicated that Cys-43 and Cys-46 are palmitoylated. The loss of palmitoylation on Lcb4 caused several effects, including mislocalization of the protein to the cytosol, reduced phosphorylation, and loss of downregulation during the stationary phase. Although Akr2 is highly homologous to Akr1, the deletion of AKR2 did not result in any remarkable phenotypes. However, overproduction of Akr2 resulted in reduced amounts of Lcb4. We demonstrated that Akr2 is an unstable protein and is degraded in the vacuole. Akr2 exhibits high affinity for Lcb4, and in Akr2-overproducing cells this interaction caused unusual delivery of Lcb4 to the vacuole and degradation