Sptlc1 is essential for myeloid differentiation and hematopoietic homeostasis

Serine palmitoyltransferase (SPT) long-chain base subunit 1 (SPTLC1) is 1 of the 2 main catalytic subunits of the SPT complex, which catalyzes the first and rate-limiting step of sphingolipid biosynthesis. Here, we show that Sptlc1 deletion in adult bone marrow (BM) cells results in defective myeloid differentiation. In chimeric mice from noncompetitive BM transplant assays, there was an expansion of the Lin- c-Kit+ Sca-1+ compartment due to increased multipotent progenitor production, but myeloid differentiation was severely compromised. We also show that defective biogenesis of sphingolipids in the endoplasmic reticulum (ER) leads to ER stress that affects myeloid differentiation. Furthermore, we demonstrate that transient accumulation of fatty acid, a substrate for sphingolipid biosynthesis, could be partially responsible for the ER stress. Independently, we find that ER stress in general, such as that induced by the chemical thapsigargin or the fatty acid palmitic acid, compromises myeloid differentiation in culture. These results identify perturbed sphingolipid metabolism as a source of ER stress, which may produce diverse pathological effects related to differential cell-type sensitivity

Phosphatidic acid phospholipase A1 mediates ER-Golgi transit of a family of G protein-coupled receptors

The coat protein II (COPII)-coated vesicular system transports newly synthesized secretory and membrane proteins from the endoplasmic reticulum (ER) to the Golgi complex. Recruitment of cargo into COPII vesicles requires an interaction of COPII proteins either with the cargo molecules directly or with cargo receptors for anterograde trafficking. We show that cytosolic phosphatidic acid phospholipase A1 (PAPLA1) interacts with COPII protein family members and is required for the transport of Rh1 (rhodopsin 1), an N-glycosylated G protein-coupled receptor (GPCR), from the ER to the Golgi complex. In papla1 mutants, in the absence of transport to the Golgi, Rh1 is aberrantly glycosylated and is mislocalized. These defects lead to decreased levels of the protein and decreased sensitivity of the photoreceptors to light. Several GPCRs, including other rhodopsins and Bride of sevenless, are similarly affected. Our findings show that a cytosolic protein is necessary for transit of selective transmembrane receptor cargo by the COPII coat for anterograde trafficking

Oleosin Is Bifunctional Enzyme That Has Both Monoacylglycerol Acyltransferase and Phospholipase Activities

In plants, fatty oils are generally stored in spherical intracellular organelles referred to as oleosomes that are covered by proteins such as oleosin. Seeds with high oil content have more oleosin than those with low oil content. However, the exact role of oleosin in oil accumulation is thus far unclear. Here, we report the isolation of a catalytically active 14 S multiprotein complex capable of acylating monoacylglycerol from the microsomal membranes of developing peanut cotyledons. Microsomal membranes from immature peanut seeds were solubilized using 8 M urea and 10 mM CHAPS. Using two-dimensional gel electrophoresis and mass spectrometry, we identified 27 proteins in the 14 S complex. The major proteins present in the 14 S complex are conarachin, the major allergen Ara h 1, and other seed storage proteins. We identified oleosin 3 as a part of the 14 S complex, which is capable of acylating monoacylglycerol. The recombinant OLE3 microsomes from Saccharomyces cerevisiae have been shown to have both a monoacylglycerol acyltransferase and a phospholipase A(2) activity. Overexpression of the oleosin 3 (OLE3) gene in S. cerevisiae resulted in an increased accumulation of diacylglycerols and triacylglycerols and decreased phospholipids. These findings provide a direct role for a structural protein (OLE3) in the biosynthesis and mobilization of plant oils

Ceramide transfer protein deficiency compromises organelle function and leads to senescence in primary cells.

Ceramide transfer protein (CERT) transfers ceramide from the endoplasmic reticulum (ER) to the Golgi complex. Its deficiency in mouse leads to embryonic death at E11.5. CERT deficient embryos die from cardiac failure due to defective organogenesis, but not due to ceramide induced apoptotic or necrotic cell death. In the current study we examined the effect of CERT deficiency in a primary cell line, namely, mouse embryonic fibroblasts (MEFs). We show that in MEFs, unlike in mutant embryos, lack of CERT does not lead to increased ceramide but causes an accumulation of hexosylceramides. Nevertheless, the defects due to defective sphingolipid metabolism that ensue, when ceramide fails to be trafficked from ER to the Golgi complex, compromise the viability of the cell. Therefore, MEFs display an incipient ER stress. While we observe that ceramide trafficking from ER to the Golgi complex is compromised, the forward transport of VSVG-GFP protein is unhindered from ER to Golgi complex to the plasma membrane. However, retrograde trafficking of the plasma membrane-associated cholera toxin B to the Golgi complex is reduced. The dysregulated sphingolipid metabolism also leads to increased mitochondrial hexosylceramide. The mitochondrial functions are also compromised in mutant MEFs since they have reduced ATP levels, have increased reactive oxygen species, and show increased glutathione reductase activity. Live-cell imaging shows that the mutant mitochondria exhibit reduced fission and fusion events. The mitochondrial dysfunction leads to an increased mitophagy in the CERT mutant MEFs. The compromised organelle function compromise cell viability and results in premature senescence of these MEFs

CERT deficiency leads to incipient ER stress in <i>Cert^gt/gt</i> MEFs.

<p>(A) The total ER ceramide levels are slightly decreased while (B) hexosylceramide levels are increased in <i>Cert^gt/gt</i> compared to the control <i>Cert^+/+</i> MEFs. (C) Basal PDI levels are increased in the mutant MEFs. Upon serum starvation the levels of IRE1α and BiP are increased in the mutant cells. (D) The ER was labeled with ER tracker green. The ER in the mutant show altered morphology and altered dynamics (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092142#pone.0092142.s002" target="_blank">videos S1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092142#pone.0092142.s003" target="_blank">S2</a>). (E). Metabolic labeling for phospholipids were performed using ³²P-orthophosphoric agent as a substrate. No visible difference in major phospholipids was observed between the wild type control and the mutant cells. (F) qPCR analysis of transcripts for GLUCS and GALCS shows increased transcript levels in P5 <i>Cert^gt/gt</i> compared to the <i>Cert^+/+</i>MEFs.</p

Altered Golgi dynamics in <i>Cert^gt/gt</i> MEFs.

<p>(A) The Golgi architecture seems to be normal as evidenced by immunofluorescence staining using a Golgi marker (GM130) and (B) The ultrastructure of Golgi seems apparently normal although we see a slightly more fragmented pattern of Golgi compared to the wild type. N- denotes the nucleus and the arrows indicated the Golgi cisternae. (C) The MEFs were incubated with DMB-C5-Cer as described under <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092142#s2" target="_blank">materials and methods</a>. While perinuclear concentration of the ceramide is clearly visible in <i>Cert^+/+</i> the <i>Cert^gt/gt</i> show a diffuse staining indicating a lack of transport of ceramide from the ER to the Golgi complex. (D) Recycling of cargo protein cholera toxin B (CTxB, associated with lipid rafts) between the plasma membrane and the Golgi complex is impaired in the mutant MEFs. (E) Quantification of the extent of recovery after photobleaching.</p