Autophagy Competes for a Common Phosphatidylethanolamine Pool with Major Cellular PE-Consuming Pathways in Saccharomyces cerevisiae

Abstract

Autophagy is a highly regulated pathway that selectively degrades cellular constituents such as protein aggregates and excessive or damaged organelles. This transport route is characterized by engulfment of the targeted cargo by autophagosomes. The formation of these double-membrane vesicles requires the covalent conjugation of the ubiquitin-like protein Atg8 to phosphatidylethanolamine (PE). However, the origin of PE and the regulation of lipid flux required for autophagy remain poorly understood. Using a genetic screen, we found that the temperature-sensitive growth and intracellular membrane organization defects of mcd4-174 and mcd4-P301L mutants are suppressed by deletion of essential autophagy genes such as ATG1 or ATG7. MCD4 encodes an ethanolamine phosphate transferase that uses PE as a precursor for an essential step in the synthesis of the glycosylphosphatidylinositol (GPI) anchor used to link a subset of plasma membrane proteins to lipid bilayers. Similar to the deletion of CHO2, a gene encoding the enzyme converting PE to phosphatidylcholine (PC), deletion of ATG7 was able to restore lipidation and plasma membrane localization of the GPIanchored protein Gas1 and normal organization of intracellular membranes. Conversely, overexpression of Cho2 was lethal in mcd4-174 cells grown at restrictive temperature. Quantitative lipid analysis revealed that PE levels are substantially reduced in the mcd4-174 mutant but can be restored by deletion of ATG7 or CHO2. Taken together, these data suggest that autophagy competes for a common PE pool with major cellular PE-consuming pathways such as the GPI anchor and PC synthesis, highlighting the possible interplay between these pathways and the existence of signals that may coordinate PE flux. KEYWORDS autophagy; Atg8; phospholipids; GPI anchor; Mcd4 L OCAL glycerophospholipid levels depend on several processes, including biosynthesis, remodeling, degradation, and interorganellar trafficking. Phosphatidylethanolamine (PE) and phosphatidylcholine (PC) are key building blocks of membrane bilayers and account for up to 50% of phospholipids in eukaryotic cells. PE is typically synthesized from phosphatidylserine (PS) by phosphatidylserine decarboxylases (Psd1 and Psd2) and can be converted to PC through the addition of three methyl groups by the methyltransferases Cho2 and Opi3 (Mcgraw and Henry 1989). Cho2 is required for the first methylation reaction during PC biosynthesis, and Opi3 catalyzes the last two steps (Carman and Han 2011). PE also can be generated through the Kennedy (or salvage) pathway from diacylglycerol and ethanolamine by the sequential action of Eki1, Ect1, and Ept1 (Henry et al. 2012;McMaster and Bell 1994). In addition to serving as main constituents of membrane bilayers, PE is also used to covalently modify a subset of proteins, thereby regulating several cellular pathways, including glycosylphosphatidylinositol (GPI) anchoring and autophagy. However, little is known about how eukaryotic cells coordinate PE homeostasis and distribution into...

Mitochondrial phosphatidylserine decarboxylase 1 (Psd1) is involved in nitrogen starvation-induced mitophagy in yeast

“…Bürgermeister et al also observed that a certain portion of PE synthesized by Psd2 can be imported into mitochondria with a moderate efficiency (Bürgermeister et al, 2004). In addition, some data have suggested that autophagy competes for a common PE pool with major cellular PE-consuming pathways, such as glycosylphosphatidylinositol anchoring (Wilson-Zbinden et al, 2015). Considering lipid trafficking and the major role of autophagy, our results are therefore not so surprising.…”

Phosphatidylethanolamine Metabolism in Health and Disease

“…Overexpression of Pem1p is lethal to the mcd4-ts yeast strain presumably by increasing the conversion of PE to PC, thus reducing the pool of PE available for GPI anchor formation. Consistent with this idea, reducing cellular PE levels by deleting PSD2 or disrupting the CDP-ethanolamine pathway also impairs growth of the mcd4-ts yeast strain (Wilson-Zbinden et al, 2015). The availability of PE as a substrate for these processes is a potentially limiting factor that may regulate flux through each pathway.…”

“…Further, PE produced by either the CDP-ethanolamine or decarboxylation pathways can be converted to PC (Kennedy and Weiss, 1956; Ridgway and Vance, 1987). PE is also a critical substrate for at least two fundamental posttranslational modifications, GPI anchors (discussed later; Menon and Stevens, 1992; Wilson-Zbinden et al, 2015) and lipidation of Atg8p/LC-3 (discussed in Section 4.4; Ichimura et al, 2000). …”

“…Growth of a yeast strain harboring a temperature sensitive ( ts ) allele of Mcd4p (Gaynor et al, 1999), a protein that appends a phosphoethanolamine group to the GPI anchor glycan core (Hong et al, 1999), is rescued at the nonpermissive temperature when genes encoding the PE-methylation enzyme, Pem1p, or either Atg7p or Atg14p, are deleted (Wilson-Zbinden et al, 2015). A decrease in Mcd4p activity results in accumulation of GPI-anchored proteins in the ER.…”

Generating anchors only to lose them: The unusual story of glycosylphosphatidylinositol anchor biosynthesis and remodeling in yeast and fungi

“…Independent of its role in GPI biosynthesis, Mcd4 is implicated in transport‐dependent metabolism of phosphatidylserine and non‐mitochondrial ATP transport across membranes . Furthermore, Mcd4 uses EtnP from a common pool of PE which is also used in the biosynthesis of phosphatidylcholine and in the formation of autophagosomes . Not surprisingly, deleting CHO2 (PE‐ N‐ methyltransferase) or ATG7 (an autophagy related gene) rescues the mcd4 mutant while simultaneously restoring lipidation and proper membrane localization of Gas1, a GPI‐AP .…”