Quality-controlled ceramide-based GPI-anchored protein sorting into selective ER exit sites.

Glycosylphosphatidylinositol-anchored proteins (GPI-APs) exit the endoplasmic reticulum (ER) through a specialized export pathway in the yeast Saccharomyces cerevisiae. We have recently shown that a very-long acyl chain (C26) ceramide present in the ER membrane drives clustering and sorting of GPI-APs into selective ER exit sites (ERES). Now, we show that this lipid-based ER sorting also involves the C26 ceramide as a lipid moiety of GPI-APs, which is incorporated into the GPI anchor through a lipid-remodeling process after protein attachment in the ER. Moreover, we also show that a GPI-AP with a C26 ceramide moiety is monitored by the GPI-glycan remodelase Ted1, which, in turn, is required for receptor-mediated export of GPI-APs. Therefore, our study reveals a quality-control system that ensures lipid-based sorting of GPI-APs into selective ERESs for differential ER export, highlighting the physiological need for this specific export pathway.

Effectiveness of dental checkups incorporating tooth brushing instruction.

The purpose of this study was to compare the effectiveness of dental checkups incorporating tooth-brushing instruction (TBI) with that of conventional dental checkups. A team consisting of one dentist and three dental hygienists saw an average of 60 employees per day on-site at an airline company. The patient's teeth were stained with a disclosing tablet and the results recorded on a Plaque Control Record (PCR) chart. The patient was then given TBI. After recording the relevant data, including TBI given and PCR scores, the charts were stored. Checkups were performed in a total of 3,854 patients between 2001 and 2005 and changes in annual scores investigated. In addition, annual shifts in mean score in patients receiving checkups over all five years were compared with those in patients receiving checkups for the first time in each of the five years. The mean score in patients receiving a checkup in 2001 was 35.1%, declining by 2.6 points to 32.5% in 2005. Among patients receiving checkups over all five years, the mean score in 2001 was 34.0%, declining by 11.2 points to 22.8% in 2005. Over the five-year period, the mean score in patients receiving checkups was 34.1%. In patients receiving checkups over all five years, the proportion with PCR scores <30% increased each year. This was because the number of patients with PCR scores ≥60% decreased each year. These findings suggest that TBI is effective in reducing poor plaque control. When compared with in patients who had not received TBI, five consecutive years of checkups was clearly effective. These results indicate that checkups incorporating TBI are more effective than conventional dental checkups that simply check for caries. In future, this type of checkup should contribute to improved preventative dentistry with minimal intervention.

Cold-sensitive phenotypes of a yeast null mutant of ARV1 support its role as a GPI flippase.

Glycosylphosphatidylinositol (GPI) is synthesized in the endoplasmic reticulum (ER) and added onto proteins to form GPI-anchored proteins. Among the many proteins involved in this process, ACAT-related enzyme-2 required for viability 1 (Arv1) is a candidate, functioning as a flippase that translocates GPI intermediates from the cytoplasmic side into the luminal side of the ER membranes. Here, we show that the deletion of the ARV1 gene in yeast leads to cold-sensitive defects in cell growth and GPI anchor synthesis. Furthermore, complementation assays show that the overexpression of a missense human ARV1-G189R mutant does not completely restore the cold-sensitive phenotypes of the yeast arv1 mutant. Our results support the proposed role of Arv1 in GPI anchor synthesis and suggest that ARV1-linked human diseases result from defective GPI anchor synthesis.

Ceramide chain length-dependent protein sorting into selective endoplasmic reticulum exit sites.

Protein sorting in the secretory pathway is crucial to maintain cellular compartmentalization and homeostasis. In addition to coat-mediated sorting, the role of lipids in driving protein sorting during secretory transport is a longstanding fundamental question that still remains unanswered. Here, we conduct 3D simultaneous multicolor high-resolution live imaging to demonstrate in vivo that newly synthesized glycosylphosphatidylinositol-anchored proteins having a very long chain ceramide lipid moiety are clustered and sorted into specialized endoplasmic reticulum exit sites that are distinct from those used by transmembrane proteins. Furthermore, we show that the chain length of ceramide in the endoplasmic reticulum membrane is critical for this sorting selectivity. Our study provides the first direct in vivo evidence for lipid chain length-based protein cargo sorting into selective export sites of the secretory pathway.

Sphingolipid/Pkh1/2-TORC1/Sch9 Signaling Regulates Ribosome Biogenesis in Tunicamycin-Induced Stress Response in Yeast.

Reduced ribosome biogenesis in response to environmental conditions is a key feature of cell adaptation to stress. For example, ribosomal genes are transcriptionally repressed when cells are exposed to tunicamycin, a protein glycosylation inhibitor that induces endoplasmic reticulum stress and blocks vesicular trafficking in the secretory pathway. Here, we describe a novel regulatory model, in which tunicamycin-mediated stress induces the accumulation of long-chain sphingoid bases and subsequent activation of Pkh1/2 signaling, which leads to decreased expression of ribosomal protein genes via the downstream effectors Pkc1 and Sch9. Target of rapamycin complex 1 (TORC1), an upstream activator of Sch9, is also required. This pathway links ribosome biogenesis to alterations in membrane lipid composition under tunicamycin-induced stress conditions. Our results suggest that sphingolipid/Pkh1/2-TORC1/Sch9 signaling is an important determinant for adaptation to tunicamycin-induced stress.