No abstract

The Saccharomyces cerevisiae NTE1 gene encodes an evolutionarily conserved phospholipase B localized to the endoplasmic reticulum (ER) that degrades phosphatidylcholine (PC) generating glycerophosphocholine and free fatty acids. We show here that the activity of NTE1-encoded phospholipase B (Nte1p) prevents the attenuation of transcription of genes encoding enzymes involved in phospholipid synthesis in response to increased rates of PC synthesis by affecting the nuclear localization of the transcriptional repressor Opi1p. Nte1p activity becomes necessary for cells growing in inositol-free media under conditions of high rates of PC synthesis elicited by the presence of choline at 37°C. The specific choline transporter encoded by the HNM1 gene is necessary for the burst of PC synthesis observed at 37°C as follows: (i) Nte1p is dispensable in an hnm1⌬ strain under these conditions, and (ii) there is a 3-fold increase in the rate of choline transport via the Hnm1p choline transporter upon a shift to 37°C. Overexpression of NTE1 alleviated the inositol auxotrophy of a plethora of mutants, including scs2⌬, scs3⌬, ire1⌬, and hac1⌬ among others. Overexpression of NTE1 sustained phospholipid synthesis gene transcription under conditions that normally repress transcription. This effect was also observed in a strain defective in the activation of free fatty acids for phosphatidic acid synthesis. No changes in the levels of phosphatidic acid were detected under conditions of altered expression of NTE1. Consistent with a synthetic impairment between challenged ER function and inositol deprivation, increased expression of NTE1 improved the growth of cells exposed to tunicamycin in the absence of inositol. We describe a new role for Nte1p toward membrane homeostasis regulating phospholipid synthesis gene transcription. We propose that Nte1p activity, by controlling PC abundance at the ER, affects lateral membrane packing and that this parameter, in turn, impacts the repressing transcriptional activity of Opi1p, the main regulator of phospholipid synthesis gene transcription.

1. Quantification of fibrosis is a key parameter in the assessment of the severity of cardiovascular disease and efficacy of future candidate therapies. Computer-assisted methods are frequently used to assess cardiac fibrosis in several experimental models. A brief survey indicated that there is a clear dearth of literature outlining detailed methodologies for computer-based assessment of cardiac fibrosis. The purpose of the present study was to provide a reliable method for a systematic assessment of cardiac fibrosis. 2. We induced cardiac fibrosis by isoproterenol (ISO) infusion in adult CD1 male mice and quantified fibrosis using a recently developed colour-subtractive computer-assisted image analysis (CS-CAIA) technique. Here, we provided a detailed description of our methodology to facilitate its wider use by other researchers. 3. We showed that the severity of ISO-induced cardiac fibrosis was similar in the apex, mid-ventricular ring and base of the adult CD1 mouse heart. In contrast with other species, such as rats and dogs, we found that uniform expression of beta(1)-adrenoceptors between different regions in CD1 mouse hearts correlated well with uniform induction of cardiac fibrosis. 4. A previous study found a negative correlation between levels of myocardial fibrosis and the degree of cardiac hypertrophy in ISO-treated Wistar rats. In contrast, we found a similar degree of cardiac fibrosis in our ISO-treated CD1 mice. 5. Our results suggest that CD1 mice are an ideal model system to study catecholamine-induced cardiac remodelling, as well as to screen candidate antifibrotic agents for future therapies.

Phosphatidylcholine is the major phospholipid in eukaryotic cells. There are two main pathways for the synthesis of phosphatidylcholine: the CDP-choline pathway present in all eukaryotes and the phosphatidylethanolamine methylation pathway present in mammalian hepatocytes and some single celled eukaryotes, including the yeast Saccharomyces cerevisiae. In S. cerevisiae, the rate-determining step in the synthesis of phosphatidylcholine via the CDP-choline pathway is catalyzed by Pct1. Pct1 converts phosphocholine and CTP to CDP-choline and pyrophosphate. In this study, we determined that Pct1 is in the nucleoplasm and at endoplasmic reticulum and nuclear membranes. Pct1 directly interacts with the ␣-importin Kap60 via a bipartite basic region in Pct1, and this region of Pct1 was required for its entry into the nucleus. Pct1 also interacted with the ␤-importin Kap95 in cell extracts, implying a model whereby Pct1 interacts with Kap60 and Kap95 with this tripartite complex transiting the nuclear pore. Exclusion of Pct1 from the nucleus by elimination of its nuclear localization signal or by decreasing Kap60 function did not affect the level of phosphatidylcholine synthesis. Diminution of Kap95 function resulted in almost complete ablation of phosphatidylcholine synthesis under conditions where Pct1 was extranuclear. The ␤-importin Kap95 is a direct regulator membrane synthesis.

Background: Barth syndrome is an inherited cardiomyopathy due to mutations in the TAZ gene. Results: A screen using taz1⌬ yeast cells identified genes whose deletion aggravated its fitness. Conclusion:The protease Yme1 is required for efficient mitophagy in the absence of TAZ1. Significance: This is the first study linking mitochondrial quality control to mitophagy as important in cells lacking TAZ1 function.