The Phosphatidylglycerol/Cardiolipin Biosynthetic Pathway Is Required for the Activation of Inositol Phosphosphingolipid Phospholipase C, Isc1p, during Growth of Saccharomyces cerevisiae

Abstract

Inositolsphingolipid phospholipase C (Isc1p) is theCeramide is a bioactive lipid that in eukaryotic cells functions as a mediator of a variety of extracellular signals through the regulation of several downstream effectors which in turn control basic cellular functions such as cell growth, cell cycle arrest, apoptosis, and senescence (1-6). Sphingomyelinases, which hydrolyze the phosphodiester linkage of sphingomyelin (SM) 1 to produce ceramide and phosphorylcholine, function as key regulators of the intracellular levels of ceramide in mammalian cells.Sphingolipids are also important regulatory molecules in yeast where they are known to be required for viability (7), optimal life span (8), cell cycle regulation (9), endocytosis (10), and regulation of responses of yeast cells to stress (1, 11). Although Saccharomyces cerevisiae do not contain SM, they do contain inositol phosphoceramides, and recently we identified a homologue of neutral sphingomyelinase in S. cerevisiae, Isc1p, which acts on phosphoceramides to generate ceramide. This enzyme displays ϳ30% identity to neutral sphingomyelinase 2 and shares with it several common features (12) including hydrolytic activity on SM, the requirement of Mg 2ϩ for optimal activity, optimal neutral pH, and the presence of a newly discovered domain that is conserved in the entire family of sphingomyelinases, the P-loop-like domain (13), which appears to be important for substrate binding and/or catalysis.In addition, both enzymes demonstrate an absolute requirement for anionic phospholipids for in vitro activation. Thus, Isc1p is activated selectively in vitro by cardiolipin (CL), phosphtidylglycerol (PG), or phosphatidylserine. In a recent study we demonstrated that the enzyme binds these anionic phospholipids, and we identified the second transmembrane domain and the C terminus of Isc1p as required for this binding, and it was proposed that this interaction plays a critical role in enzyme function through a novel tethering mechanism of enzyme activation by lipid cofactors (14).Given these specific requirements for anionic phospholipids in vitro, it became important to determine whether the enzyme requires phospholipids for activation in vivo. Our attention was particularly directed to the possible roles of PG and CL as the deletion of PGS1, the gene that encodes the enzyme responsible for the synthesis of PG phosphate, and subsequent synthesis of PG and CL was shown to cause a late defect during growth in glucose media similar to that of ISC1. Moreover, these lipids are almost exclusively present in mitochondria, and we have shown recently that Isc1p localizes preferentially to mitochondria in the post-diauxic phase of growth.Synthesis of CL in yeast requires three sequential reactions.

Thematic Review Series: Sphingolipids. New insights into sphingolipid metabolism and function in budding yeast

“…Isc1 activity is required for growth on a nonfermentable carbon source such as during the diauxic shift, implying a role in respiration/mitochondria (56). A mitochondrial connection is supported by the observation that Isc1 moves from the ER to the mitochondria as cells progress from fermentative to respiratory growth during the diauxic shift (57).…”

“…During this time, the specific activity of the enzyme increases by 3-to 5-fold and phytoceramide increases by 4-fold, but only if Isc1 is active. Lipids enriched in mitochondria, phosphatidylglycerol, and cardiolipin activate and appear to be physiologically important regulators of enzyme activity (56). Isc1 in cells grown to saturation (postdiauxic shift) was recently shown to reside in the outer leaflet of the mitochondrial membrane (58).…”

Isc1p Plays a Key Role in Hydrogen Peroxide Resistance and Chronological Lifespan through Modulation of Iron Levels and Apoptosis

“…This is consistent with a previous report showing that the S. pombe Isc1p homologue, Ccs1p, is important for cell wall organization as it coordinates cell wall formation and division (Feoktistova et al, 2001). The induction of the glycolytic pathway probably aims to compensate for a decreased production of ATP in the mitochondria, as isc1⌬ mutants have a decreased capacity to grow by respiration (Vaena de Avalos et al, 2005). In agreement with the increased expression of four genes encoding components of the nonreductive iron uptake system (ARN1, ARN2, ARN3, and ARN4), and two genes encoding cell wall mannoproteins involved in the retention of siderophore-iron in the cell wall (FIT2 and FIT3; Philpott et al, 2002), iron levels increased.…”

“…The activation of Isc1p is dependent on Pgs1p, which catalyzes the committed step in the synthesis of phosphatidylglycerol and cardiolipin in the mitochondria. Loss of either ISC1 or PGS1 results in down-regulation of the mitochondrial cytochrome c oxidase subunits Cox3p and Cox4p, suggesting that Isc1p mediates at least some functions downstream of phosphatidylglycerol/cardiolipin (Ostrander et al, 2001;Vaena de Avalos et al, 2005).…”

Thematic Review Series: Sphingolipids. ISC1 (inositol phosphosphingolipid-phospholipase C), the yeast homologue of neutral sphingomyelinases

“…A major finding of this study was that Isc1p localized in the endoplasmic reticulum (ER) during the prediauxic phase and in the mitochondria during the postdiauxic phase (8). A substantial growth defect of isc1D strains when grown on a nonfermentable carbon source (NFCS) such us glycerol was described in a subsequent study (17).…”