Maintenance of sphingolipid homeostasis is critical for cell growth and programmed cell death (PCD). Serine palmitoyltransferase (SPT), composed of LCB1 and LCB2 subunits, catalyzes the primary regulatory point for sphingolipid synthesis. Small subunits of SPT (ssSPT) that strongly stimulate SPT activity have been identified in mammals, but the role of ssSPT in eukaryotic cells is unclear. Candidate Arabidopsis thaliana ssSPTs, ssSPTa and ssSPTb, were identified and characterized. Expression of these 56-amino acid polypeptides in a Saccharomyces cerevisiae SPT null mutant stimulated SPT activity from the Arabidopsis LCB1/LCB2 heterodimer by >100-fold through physical interaction with LCB1/LCB2. ssSPTa transcripts were more enriched in all organs and >400-fold more abundant in pollen than ssSPTb transcripts. Accordingly, homozygous ssSPTa T-DNA mutants were not recoverable, and 50% nonviable pollen was detected in heterozygous ssspta mutants. Pollen viability was recovered by expression of wild-type ssSPTa or ssSPTb under control of the ssSPTa promoter, indicating ssSPTa and ssSPTb functional redundancy. SPT activity and sensitivity to the PCD-inducing mycotoxin fumonisin B 1 (FB 1 ) were increased by ssSPTa overexpression. Conversely, SPT activity and FB 1 sensitivity were reduced in ssSPTa RNA interference lines. These results demonstrate that ssSPTs are essential for male gametophytes, are important for FB 1 sensitivity, and limit sphingolipid synthesis in planta.

Sphingolipids are membrane and bioactive lipids that are required for many aspects of normal mammalian development and physiology. However, the importance of the regulatory mechanisms that control sphingolipid levels in these processes is not well understood. The mammalian ORMDL proteins (ORMDL1, 2 and 3) mediate feedback inhibition of the de novo synthesis pathway of sphingolipids by inhibiting serine palmitoyl transferase in response to elevated ceramide levels. To understand the function of ORMDL proteins in vivo, we studied mouse knockouts (KOs) of the Ormdl genes. We found that Ormdl1 and Ormdl3 function redundantly to suppress the levels of bioactive sphingolipid metabolites during myelination of the sciatic nerve. Without proper ORMDL-mediated regulation of sphingolipid synthesis, severe dysmyelination results. Our data indicate that the Ormdls function to restrain sphingolipid metabolism in order to limit levels of dangerous metabolic intermediates that can interfere with essential physiological processes such as myelination.

Journal of Lipid Research Volume 55, 20142521 the metabolic pathway ( 3 ). Ceramide undergoes anabolic reactions to generate sphingomyelin and various glycosphingolipids, or catabolic reactions, which lead to the generation of sphingosine and sphingosine-1-phosphate (S1P). Alterations in ceramide metabolism have been implicated in many pathophysiologies, including aging ( 4-6 ), neurodegeneration ( 7,8 ), metabolic diseases ( 9-15 ), cancer (16)(17)(18)(19)(20), and stress responses ( 5 ). However, the mechanisms that regulate cellular ceramide levels under physiologic and pathophysiologic conditions are still not well-understood.Changes in the levels of ceramide and other sphingolipid metabolites have been shown to affect macroautophagy (referred to hereafter as autophagy) in a variety of cell types ( 21-27 ). Autophagy is a catabolic process that starts with the generation of a double-membrane cup-like phagophore from the ER or other sources ( 28, 29 ); the phagophore then captures cellular material and matures into an autophagosome that will subsequently fuse with a lysosome to form an autolysosome, enabling degradation of the engulfed material. This process is crucial for removal of pathogens and damaged proteins and organelles, as well as for the reutilization of nutrients to generate energy and maintain homeostasis. Clearance of toxic or defective cellular components protects from degenerative, metabolic, and infl ammatory diseases ( 30 ). Some forms of autophagy are specifi c, uniquely targeting mitochondria (mitophagy) ( 31 ), segments of the ER (ER-phagy or reticulophagy) ( 32 ), or triglyceride stores (lipophagy) ( 33 ) for degradation. Impaired autophagy is encountered, along with increased ceramide levels, in a number of pathophysiologic conditions, including aging ( 34, 35 ), neurodegeneration ( 36, 37 ), obesity ( 10 ), and type 2 diabetes ( 9 ).Abstract Sphingolipid levels are tightly regulated to maintain cellular homeostasis. During pathologic conditions such as in aging, infl ammation, and metabolic and neurodegenerative diseases, levels of some sphingolipids, including the bioactive metabolite ceramide, are elevated. Sphingolipid metabolism has been linked to autophagy, a critical catabolic process in both normal cell function and disease; however, the in vivo relevance of the interaction is not wellunderstood. Here, we show that blocking autophagy in the liver by deletion of the Atg7 gene, which is essential for autophagosome formation, causes an increase in sphingolipid metabolites including ceramide. We also show that overexpression of serine palmitoyltransferase to elevate de novo sphingolipid biosynthesis induces autophagy in the liver. The results reveal autophagy as a process that limits excessive ceramide levels and that is induced by excessive elevation of de novo sphingolipid synthesis in the liver. Dysfunctional autophagy may be an underlying mechanism causing elevations in ceramide that may contribute to pathogenesis in diseases. Sphingolipids are a structurally and functionally di...