A Genome-Wide Screen Reveals That Endocytic Genes Are Important for Pma1p Asymmetry during Cell Division in Saccharomyces cerevisiae.

An asymmetry in cytosolic pH between mother and daughter cells was reported to underlie cellular aging in the budding yeast Saccharomyces cerevisiae; however, the underlying mechanism remains unknown. Preferential accumulation of Pma1p, which pumps cytoplasmic protons out of cells, at the plasma membrane of mother cells, but not of their newly-formed daughter cells, is believed to be responsible for the pH increase in mother cells by reducing the level of cytoplasmic protons. This, in turn, decreases the acidity of vacuoles, which is well correlated with aging of yeast cells. In this study, to identify genes that regulate the preferential accumulation of Pma1p in mother cells, we performed a genome-wide screen using a collection of single gene deletion yeast strains. A subset of genes involved in the endocytic pathway, such as VPS8, VPS9, and VPS21, was important for Pma1p accumulation. Unexpectedly, however, there was little correlation between deletion of each of these genes and the replicative lifespan of yeast, suggesting that Pma1p accumulation in mother cells is not the key determinant that underlies aging of mother cells.

Ergosterol interacts with Sey1p to promote atlastin-mediated endoplasmic reticulum membrane fusion in Saccharomyces cerevisiae.

Sterols play critical roles in various membrane fusion events, including soluble NSF attachment protein receptor-mediated membrane fusion, mainly by modulating the physical properties of biologic membranes; however, it remains unclear whether they also function in atlastin-mediated endoplasmic reticulum (ER) membrane fusion. Although ergosterol, the major sterol in yeast, is essential for fusion of Sey1p (yeast atlastin)-containing liposomes with an ER-mimicking lipid composition, fusion of phosphatidylcholine/phosphatidylserine liposomes does not require sterols. Here, we examined whether sterols are important for Sey1p-mediated ER fusion in Saccharomyces cerevisiae using an in vitro ER fusion assay with isolated yeast ER microsomes. Ergosterol-specific ligands inhibited microsome fusion, indicating that ergosterol is critical for ER fusion. However, microsomes isolated from yeast strains lacking genes that encode enzymes involved in synthesis of ergosterol from lanosterol still fused, suggesting that other sterols can replace ergosterol and support Sey1p-mediated ER fusion. Importantly, disruption of sterol-binding motifs in the transmembrane regions of Sey1p markedly reduced ER fusion. Sey1p physically interacted with Erg11p and Erg4p, which function in ergosterol biosynthesis, suggesting that Sey1p recruits ergosterol-synthesizing enzymes to fusion sites and thereby enriches ergosterol, which, in turn, may recruit more Sey1p. This positive feedback loop may facilitate ER membrane fusion by concentrating fusion factors at fusion sites.-Lee, M., Moon, Y., Lee, S., Lee, C., Jun, Y. Ergosterol interacts with Sey1p to promote atlastin-mediated endoplasmic reticulum membrane fusion in Saccharomyces cerevisiae.

Human atlastins are sufficient to drive the fusion of liposomes with a physiological lipid composition.

The dynamin-like GTPase atlastin is believed to be the minimal machinery required for homotypic endoplasmic reticulum (ER) membrane fusion, mainly because Drosophila atlastin is sufficient to drive liposome fusion. However, it remains unclear whether mammalian atlastins, including the three human atlastins, are sufficient to induce liposome fusion, raising doubts about their major roles in mammalian cells. Here, we show that all human atlastins are sufficient to induce fusion when reconstituted into liposomes with a lipid composition mimicking that of the ER. Although the fusogenic activity of ATL1, which is predominantly expressed in neuronal cells, was weaker than that of ATL2 or ATL3, the addition of M1-spastin, a neuron-specific factor, markedly increased ATL1-mediated liposome fusion. Although we observed efficient fusion between ER microsomes isolated from cultured, non-neuronal cells that predominantly express ATL2-1, an autoinhibited isoform of ATL2, ATL2-1 failed to support liposome fusion by itself as reported previously, indicating that cellular factors enable ATL2-1 to mediate ER fusion in vivo.

The Effects of Regulatory Lipids on Intracellular Membrane Fusion Mediated by Dynamin-Like GTPases.

Membrane fusion mediates a number of fundamental biological processes such as intracellular membrane trafficking, fertilization, and viral infection. Biological membranes are composed of lipids and proteins; while lipids generally play a structural role, proteins mediate specific functions in the membrane. Likewise, although proteins are key players in the fusion of biological membranes, there is emerging evidence supporting a functional role of lipids in various membrane fusion events. Intracellular membrane fusion is mediated by two protein families: SNAREs and membrane-bound GTPases. SNARE proteins are involved in membrane fusion between transport vesicles and their target compartments, as well as in homotypic fusion between organelles of the same type. Membrane-bound GTPases mediate mitochondrial fusion and homotypic endoplasmic reticulum fusion. Certain membrane lipids, known as regulatory lipids, regulate these membrane fusion events by directly affecting the function of membrane-bound GTPases, instead of simply changing the biophysical and biochemical properties of lipid bilayers. In this review, we provide a summary of the current understanding of how regulatory lipids affect GTPase-mediated intracellular membrane fusion by focusing on the functions of regulatory lipids that directly affect fusogenic GTPases.

Molecular mechanisms of atlastin-mediated ER membrane fusion revealed by a FRET-based single-vesicle fusion assay.

Homotypic fusion of endoplasmic reticulum membranes is driven by atlastin GTPases; however, the underlying mechanism remains largely unknown. Here, using a FRET-based single-vesicle fusion assay with liposomes bearing the yeast atlastin Sey1p, we investigated the molecular mechanisms of atlastin-mediated membrane tethering and fusion. Although Sey1p-bearing proteoliposomes frequently underwent membrane tethering in a GTP hydrolysis-dependent manner as reported in studies using bulk assays, only a small fraction of the tethered liposomes proceeded to fusion. Strikingly, the rest of the tethered liposomes failed to fuse or dissociate. This stable tethering, however, did not require continued GTP hydrolysis because GTP omission and magnesium chelation did not disrupt tethering. Interestingly, an increased Sey1p density on the membrane markedly accelerated tethering but barely affected the fusion rate of the tethered liposomes, indicating that Sey1p requires additional factors to support efficient fusion in vivo. Finally, the assay also revealed that Sey1p-mediated liposome fusion occurs through hemifusion, suggesting the mechanistic conservation between biological membrane fusion events despite the existence of diverse fusogens.

Maximal Fat Oxidation Rate during Exercise in Korean Women with Type 2 Diabetes Mellitus.

BACKGROUND: The purpose of this study was to determine the appropriate exercise intensity associated with maximum fat oxidation, improvement of body composition, and metabolic status in Korean women with type 2 diabetes mellitus (T2DM). METHODS: The study included a T2DM group (12 women) and a control group (12 women). The groups were matched in age and body mass index. The subjects performed a graded exercise test on a cycle ergometer to measure their maximal fat oxidation (Fatmax). We also measured their body composition, metabolic profiles, and mitochondrial DNA (mtDNA). RESULTS: The exercise intensity for Fatmax was significantly lower in the T2DM group (34.19% maximal oxygen uptake [VO2 max]) than the control group (51.80% VO2 max). Additionally, the rate of fat oxidation during exercise (P<0.05) and mtDNA (P<0.05) were significantly lower in the T2DM group than the control group. The VO2 max level (P<0.001) and the insulin level (P<0.05) were positively correlated with the rate of fat oxidation. CONCLUSION: The results of this study suggest lower exercise intensity that achieves Fatmax is recommended for improving fat oxidation and enhancing fitness levels in Korean women with T2DM. Our data could be useful when considering an exercise regimen to improve health and fitness.

SNAREs support atlastin-mediated homotypic ER fusion in Saccharomyces cerevisiae.

Dynamin-like GTPases of the atlastin family are thought to mediate homotypic endoplasmic reticulum (ER) membrane fusion; however, the underlying mechanism remains largely unclear. Here, we developed a simple and quantitative in vitro assay using isolated yeast microsomes for measuring yeast atlastin Sey1p-dependent ER fusion. Using this assay, we found that the ER SNAREs Sec22p and Sec20p were required for Sey1p-mediated ER fusion. Consistently, ER fusion was significantly reduced by inhibition of Sec18p and Sec17p, which regulate SNARE-mediated membrane fusion. The involvement of SNAREs in Sey1p-dependent ER fusion was further supported by the physical interaction of Sey1p with Sec22p and Ufe1p, another ER SNARE. Furthermore, our estimation of the concentration of Sey1p on isolated microsomes, together with the lack of fusion between Sey1p proteoliposomes even with a 25-fold excess of the physiological concentration of Sey1p, suggests that Sey1p requires additional factors to support ER fusion in vivo. Collectively, our data strongly suggest that SNARE-mediated membrane fusion is involved in atlastin-initiated homotypic ER fusion.

Effect on Glycemic, Blood Pressure, and Lipid Control according to Education Types.

BACKGROUND: Diabetes self-management education and reinforcement are important for effective management of the disease. We investigated the effectiveness of interactive small-group education on glycemic, blood pressure, and lipid levels. METHODS: For this study, 207 type 2 diabetes patients with suboptimal glycemic control (HbA1c levels >6.5%) were enrolled. The conventional education group received an existing education program from April to November in 2006, and the interactive education group received a new small-group education program from December 2006 to July 2007. The two groups were comparatively analyzed for changes in blood sugar, glycated hemoglobin, lipid, and blood pressure at baseline, 3, 6, and 12 months and the proportion of patients achieving target goals at 12 months. RESULTS: After 12 months of follow-up, HbA1c levels in the interactive education group were significantly lower than in the conventional education group (6.7% vs. 6.4%, P<0.001). Fasting and 2 hour postprandial glucose concentrations, total cholesterol, and low density lipoprotein cholesterol were significantly lower in the interactive education group than in the conventional education group. The proportion of patients that achieved target goals was significantly higher in the interactive education group. CONCLUSION: The small-group educational method improved and re-established the existing group educational method. This finding suggests that the importance of education appears to be related to the method by which it is received rather than the education itself. Thus, the use of small-group educational methods to supplement existing educational methods established for diverse age levels should be considered in the future.