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Orm family proteins mediate sphingolipid homeostasis.

Breslow DK, Collins SR, Bodenmiller B, Aebersold R, Simons K, Shevchenko A, Ejsing CS, Weissman JS - Nature (2010)

Bottom Line: Starting from an unbiased functional genomic approach in Saccharomyces cerevisiae, we identify Orm proteins as negative regulators of sphingolipid synthesis that form a conserved complex with serine palmitoyltransferase, the first and rate-limiting enzyme in sphingolipid production.We also define a regulatory pathway in which phosphorylation of Orm proteins relieves their inhibitory activity when sphingolipid production is disrupted.Changes in ORM gene expression or mutations to their phosphorylation sites cause dysregulation of sphingolipid metabolism.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular and Molecular Pharmacology, University of California, San Francisco, 1700 4th Street, San Francisco, California 94158, USA.

ABSTRACT
Despite the essential roles of sphingolipids both as structural components of membranes and critical signalling molecules, we have a limited understanding of how cells sense and regulate their levels. Here we reveal the function in sphingolipid metabolism of the ORM genes (known as ORMDL genes in humans)-a conserved gene family that includes ORMDL3, which has recently been identified as a potential risk factor for childhood asthma. Starting from an unbiased functional genomic approach in Saccharomyces cerevisiae, we identify Orm proteins as negative regulators of sphingolipid synthesis that form a conserved complex with serine palmitoyltransferase, the first and rate-limiting enzyme in sphingolipid production. We also define a regulatory pathway in which phosphorylation of Orm proteins relieves their inhibitory activity when sphingolipid production is disrupted. Changes in ORM gene expression or mutations to their phosphorylation sites cause dysregulation of sphingolipid metabolism. Our work identifies the Orm proteins as critical mediators of sphingolipid homeostasis and raises the possibility that sphingolipid misregulation contributes to the development of childhood asthma.

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Orm1/2 are regulated in response to disruption of sphingolipid synthesisa, Serial dilutions of the indicated strains were spotted on plates with 0, 200, or 400 ng/ml myriocin and imaged after growth for 24–48 hr. b, LCBs were extracted and analyzed from wild-type (WT) and ORM1/2 deletion strains grown in media supplemented with the indicated concentrations of myriocin. The sums of C18 dihydrosphingosine and phytosphingosine peak intensities from n ≥ 2 experiments are shown (relative to wild-type LCB levels in the absence of myriocin). c, Native immunoprecipitations of 3×Flag-tagged Orm1 and Orm2 were performed from strains grown in standard media or media supplemented with 150 ng/ml myriocin and analyzed by Western blot. The indicated strains also expressed 3×HA-Orm1/2 from their endogenous loci (diploid strains were used to examine self-association of Orm1 and Orm2). d, Lysates were prepared from yeast expressing 3×Flag-Orm1/2 after growth in media containing the indicated concentrations of myriocin. Western blots show phosphorylated forms of 3×Flag-Orm1 (P-Orm1) and 3×Flag-Orm2 (P-Orm2) after separation on phosphate-affinity gels35. An immunoprecipitated (IP) sample treated with calf intestine phosphatase (CIP) shows the position of un-phosphorylated Orm1/2.
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Figure 4: Orm1/2 are regulated in response to disruption of sphingolipid synthesisa, Serial dilutions of the indicated strains were spotted on plates with 0, 200, or 400 ng/ml myriocin and imaged after growth for 24–48 hr. b, LCBs were extracted and analyzed from wild-type (WT) and ORM1/2 deletion strains grown in media supplemented with the indicated concentrations of myriocin. The sums of C18 dihydrosphingosine and phytosphingosine peak intensities from n ≥ 2 experiments are shown (relative to wild-type LCB levels in the absence of myriocin). c, Native immunoprecipitations of 3×Flag-tagged Orm1 and Orm2 were performed from strains grown in standard media or media supplemented with 150 ng/ml myriocin and analyzed by Western blot. The indicated strains also expressed 3×HA-Orm1/2 from their endogenous loci (diploid strains were used to examine self-association of Orm1 and Orm2). d, Lysates were prepared from yeast expressing 3×Flag-Orm1/2 after growth in media containing the indicated concentrations of myriocin. Western blots show phosphorylated forms of 3×Flag-Orm1 (P-Orm1) and 3×Flag-Orm2 (P-Orm2) after separation on phosphate-affinity gels35. An immunoprecipitated (IP) sample treated with calf intestine phosphatase (CIP) shows the position of un-phosphorylated Orm1/2.

Mentions: We next explored how Orm proteins modulate sphingolipid production by identifying proteins bound by Orm1 and Orm2 in vivo. Isolation of functional 3×Flag-tagged forms of Orm1 and Orm2 expressed from their endogenous loci revealed a previously uncharacterized, roughly stoichiometric complex (Fig. 2a). Mass spectrometry showed that this complex comprises the serine palmitoyltransferase proteins Lcb1, Lcb2 and Tsc3, in addition to the phosphoinositide phosphatase Sac132 (Supplementary Table 1). To further characterize physical interactions among these proteins, we utilized a 3×Flag-tagged form of Lcb1 that is able to rescue the inviability seen upon loss of LCB1 (although this allele does exhibit mild sensitivity to myriocin). Purification of 3×Flag-Lcb1 yielded the same components seen in the Orm1/2 pull-downs (Fig. 2b), indicating that the Orm-associated proteins are likely to exist in a single complex. Orm proteins and potentially their binding partners may also form higher-order oligomers, as we detected Orm2 co-immunoprecipitation with 3×Flag-Orm1 (and vice versa) and self-association of differently tagged copies of the same Orm protein (Fig. 2a and Fig. 4c).


Orm family proteins mediate sphingolipid homeostasis.

Breslow DK, Collins SR, Bodenmiller B, Aebersold R, Simons K, Shevchenko A, Ejsing CS, Weissman JS - Nature (2010)

Orm1/2 are regulated in response to disruption of sphingolipid synthesisa, Serial dilutions of the indicated strains were spotted on plates with 0, 200, or 400 ng/ml myriocin and imaged after growth for 24–48 hr. b, LCBs were extracted and analyzed from wild-type (WT) and ORM1/2 deletion strains grown in media supplemented with the indicated concentrations of myriocin. The sums of C18 dihydrosphingosine and phytosphingosine peak intensities from n ≥ 2 experiments are shown (relative to wild-type LCB levels in the absence of myriocin). c, Native immunoprecipitations of 3×Flag-tagged Orm1 and Orm2 were performed from strains grown in standard media or media supplemented with 150 ng/ml myriocin and analyzed by Western blot. The indicated strains also expressed 3×HA-Orm1/2 from their endogenous loci (diploid strains were used to examine self-association of Orm1 and Orm2). d, Lysates were prepared from yeast expressing 3×Flag-Orm1/2 after growth in media containing the indicated concentrations of myriocin. Western blots show phosphorylated forms of 3×Flag-Orm1 (P-Orm1) and 3×Flag-Orm2 (P-Orm2) after separation on phosphate-affinity gels35. An immunoprecipitated (IP) sample treated with calf intestine phosphatase (CIP) shows the position of un-phosphorylated Orm1/2.
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Figure 4: Orm1/2 are regulated in response to disruption of sphingolipid synthesisa, Serial dilutions of the indicated strains were spotted on plates with 0, 200, or 400 ng/ml myriocin and imaged after growth for 24–48 hr. b, LCBs were extracted and analyzed from wild-type (WT) and ORM1/2 deletion strains grown in media supplemented with the indicated concentrations of myriocin. The sums of C18 dihydrosphingosine and phytosphingosine peak intensities from n ≥ 2 experiments are shown (relative to wild-type LCB levels in the absence of myriocin). c, Native immunoprecipitations of 3×Flag-tagged Orm1 and Orm2 were performed from strains grown in standard media or media supplemented with 150 ng/ml myriocin and analyzed by Western blot. The indicated strains also expressed 3×HA-Orm1/2 from their endogenous loci (diploid strains were used to examine self-association of Orm1 and Orm2). d, Lysates were prepared from yeast expressing 3×Flag-Orm1/2 after growth in media containing the indicated concentrations of myriocin. Western blots show phosphorylated forms of 3×Flag-Orm1 (P-Orm1) and 3×Flag-Orm2 (P-Orm2) after separation on phosphate-affinity gels35. An immunoprecipitated (IP) sample treated with calf intestine phosphatase (CIP) shows the position of un-phosphorylated Orm1/2.
Mentions: We next explored how Orm proteins modulate sphingolipid production by identifying proteins bound by Orm1 and Orm2 in vivo. Isolation of functional 3×Flag-tagged forms of Orm1 and Orm2 expressed from their endogenous loci revealed a previously uncharacterized, roughly stoichiometric complex (Fig. 2a). Mass spectrometry showed that this complex comprises the serine palmitoyltransferase proteins Lcb1, Lcb2 and Tsc3, in addition to the phosphoinositide phosphatase Sac132 (Supplementary Table 1). To further characterize physical interactions among these proteins, we utilized a 3×Flag-tagged form of Lcb1 that is able to rescue the inviability seen upon loss of LCB1 (although this allele does exhibit mild sensitivity to myriocin). Purification of 3×Flag-Lcb1 yielded the same components seen in the Orm1/2 pull-downs (Fig. 2b), indicating that the Orm-associated proteins are likely to exist in a single complex. Orm proteins and potentially their binding partners may also form higher-order oligomers, as we detected Orm2 co-immunoprecipitation with 3×Flag-Orm1 (and vice versa) and self-association of differently tagged copies of the same Orm protein (Fig. 2a and Fig. 4c).

Bottom Line: Starting from an unbiased functional genomic approach in Saccharomyces cerevisiae, we identify Orm proteins as negative regulators of sphingolipid synthesis that form a conserved complex with serine palmitoyltransferase, the first and rate-limiting enzyme in sphingolipid production.We also define a regulatory pathway in which phosphorylation of Orm proteins relieves their inhibitory activity when sphingolipid production is disrupted.Changes in ORM gene expression or mutations to their phosphorylation sites cause dysregulation of sphingolipid metabolism.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular and Molecular Pharmacology, University of California, San Francisco, 1700 4th Street, San Francisco, California 94158, USA.

ABSTRACT
Despite the essential roles of sphingolipids both as structural components of membranes and critical signalling molecules, we have a limited understanding of how cells sense and regulate their levels. Here we reveal the function in sphingolipid metabolism of the ORM genes (known as ORMDL genes in humans)-a conserved gene family that includes ORMDL3, which has recently been identified as a potential risk factor for childhood asthma. Starting from an unbiased functional genomic approach in Saccharomyces cerevisiae, we identify Orm proteins as negative regulators of sphingolipid synthesis that form a conserved complex with serine palmitoyltransferase, the first and rate-limiting enzyme in sphingolipid production. We also define a regulatory pathway in which phosphorylation of Orm proteins relieves their inhibitory activity when sphingolipid production is disrupted. Changes in ORM gene expression or mutations to their phosphorylation sites cause dysregulation of sphingolipid metabolism. Our work identifies the Orm proteins as critical mediators of sphingolipid homeostasis and raises the possibility that sphingolipid misregulation contributes to the development of childhood asthma.

Show MeSH
Related in: MedlinePlus