Sphingolipid synthesis is involved in autophagy in Saccharomyces cerevisiae

Abstract

In eukaryotes, autophagy is a conserved protein degradation system that degrades cytoplasmic components by encompassing them with double-membrane structures, called autophagosomes, and delivering them to the lytic compartments of vacuoles/lysosomes. Certain Atg proteins are known to be involved in autophagy, yet the identity and function of lipid molecules involved remain largely unknown. We investigated the involvement of sphingolipids in autophagy using Saccharomyces cerevisiae. Inhibiting synthesis of the simplest complex sphingolipid, inositol phosphorylceramide (IPC), resulted in reduced autophagic activities. Similar results were obtained using myriocin, an inhibitor of the first step in sphingolipid synthesis.Our results indicate that sphingolipids, especially IPC, are required for autophagy.Inhibition of sphingolipid synthesis had no effect on formation of Atg12-Atg5 or Atg8-phosphatidylethanolamine conjugates, on maturation of vacuolar proteases, or on formation of the pre-autophagosomal structure (PAS). These results suggest that sphingolipids are not involved in the cellular signaling that leads to formation of the PAS, but may be involved in the process of autophagosome formation.

Sphingolipids: regulators of crosstalk between apoptosis and autophagy

“…177 ), all or some of which may mediate autophagosome biogenesis and/or maturation. In support of this hypothesis, a recent study demonstrated that the inhibition of sphingolipid synthesis in Saccharomyces cerevisiae suppressed autophagosome biogenesis through a mechanism that was both independent of the Atg12-Atg5 and Atg8-PE conjugation systems as well as the formation of preautophagosomal structures (PAS), suggesting that sphingolipids play a critical role in the elongation, expansion, or closure of autophagosomal membranes ( 95 ). In addition, de novo ceramide synthesis is essential for autophagy following macrophage activation, and ceramide is observed to associate with autophagosomes in these cells ( 90 ).…”

“…Endogenous ceramide species are critical for the induction of autophagy, as inhibition of CerS by fumonisin B1 or SPT by myriocin completely suppresses autophagy induced in response to short-chain ceramides and tamoxifen, respectively ( 85,88 ). Consistently, de novo synthesis is reported to be essential for the induction of autophagy in activated RAW264.7 cells ( 90 ) as well as in Saccharomyces cerevisiae ( 95 ). However, the mechanism and function of ceramideinduced autophagy remains unclear.…”

Membrane Protein Rim21 Plays a Central Role in Sensing Ambient pH in Saccharomyces cerevisiae

“…Immunoblot Analysis-Proteins were separated by SDS-PAGE and transferred to Immobilon TM polyvinylidene difluoride membrane (Millipore, Billerica, MA) as described previously (29). The membrane was incubated with anti-FLAG (M2, Stratagene), anti-HA (Y-11, Santa Cruz Biotechnology, Santa Cruz, CA; or 3F10, Roche Diagnostics), or anti-Pgk1 (Molecular Probes, Eugene, OR) antibody.…”

Signaling Events of the Rim101 Pathway Occur at the Plasma Membrane in a Ubiquitination-Dependent Manner

“…Proteins were separated by SDS-PAGE and transferred to an Immobilon polyvinylidene difluoride membrane (Millipore, Billerica, MA) as described previously (24). The membrane was incubated with an anti-HA (16B12 [Covance, Princeton, NJ] or TANA2 [Medical & Biological Laboratories, Nagoya, Japan]), anti-GFP (JL-8; TaKaRa Bio, Shiga, Japan), anti-carboxypeptidase Y (anti-CPY) (Molecular Probes, Eugene, OR), anti-AID (BioROIS, Tokyo, Japan), or anti-Pgk1 (Molecular Probes) antibody.…”