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Autophagy is a degradative cellular pathway that protects eukaryotic cells from starvation/stress. Phosphatidylinositol 4-kinases, Pik1p and Stt4p, are indispensable for autophagy in budding yeast, but participation of PtdIns-4 kinases and their product, phosphatidylinositol 4-phosphate [PtdIns(4)P], is not understood. Nanoscale membrane lipid distribution analysis showed PtdIns(4)P is more abundant in yeast autophagosomes in the luminal leaflet than the cytoplasmic leaflet. PtdIns(4)P is confined to the cytoplasmic leaflet of autophagosomal inner and outer membranes in mammalian cells. Using temperature-conditional single PIK1 or STT4 PtdIns 4-kinase mutants, autophagic bodies in the vacuole of PIK1 and STT4 mutant cells dramatically decreased at restrictive temperatures, and the number of autophagosomes in the cytosol of PIK1 mutants cells was also decreased, whereas autophagosome levels of STT4 mutant cells were comparable to that of wild-type and STT4 mutant cells at permissive temperatures. Localization of PtdIns(4)P in the luminal leaflet in the biological membrane is a novel finding, and differences in PtdIns(4)P distribution suggest substantial differences between yeast and mammals. We also demonstrate in this study that Pik1p and Stt4p play essential roles in autophagosome formation and autophagosome–vacuole fusion in yeast cells, respectively. • PtdIns(4)P is localized in the luminal leaflet of the yeast autophagsomal membranes. • PtdIns(4)P localization in yeast autophagosomal membrane is opposite to mammals. • Pik1p plays essential roles in autophagosome formation. • Stt4p is important for the process of autophagosome–vacuole fusion. [ABSTRACT FROM AUTHOR]

• GM1 and GM3 are localized in the luminal leaflet of the inner membrane complex (IMC) in Toxoplasma gondii. • Raft microdomains exist in the luminal, but not cytoplasmic, leaflet in the IMC in T. gondii. • T. gondii obtains GM1 and GM3 from the infected host cell Membrane microdomains or rafts, sterol- and sphingolipid-rich microdomains in the plasma membrane have been studied extensively in mammalian cells. Recently, rafts were found to mediate virulence in a variety of parasites, including Toxoplasma gondii. However, it has been difficult to examine a two-dimensional distribution of lipid molecules at a nanometer scale. We tried to determine the distribution of glycosphingolipids GM1 and GM3, putative raft components in the T. gondii cell membrane in this study, using a rapid-frozen and freeze-fractured immuno-electron microscopy method. This method physically stabilized molecules in situ, to minimize the probability of artefactual disruption. Labeling of GM3, but not GM1, was observed in the exoplasmic (or luminal), but not the cytoplasmic, leaflet of the inner membrane complex (IMC) in T. gondii infected in human foreskin fibroblast-1 (HFF-1). No labeling was detected in any leaflet of the T. gondii plasma membrane. In contrast to HFF-1, T. gondii infected in mouse fibroblast (MF), labelings of both GM1 and GM3 were detected in the IMC luminal leaflet, although GM1′s gold labeling density was very low. The same freeze-fracture EM method showed that both GM1 and GM3 were expressed in the exoplasmic leaflet of the MF plasma membrane. However, labeling of only GM3, but not GM1, was detected in the exoplasmic leaflet of the HFF-1 plasma membrane. These results suggest that GM1 or GM3, localized in the IMC, is obtained from the plasma membranes of infected host mammalian cells. Furthermore, the localization of microdomains or rafts in the luminal leaflets of the intracellular confined space IMC organelle of T. gondii suggests a novel characteristic of rafts. [ABSTRACT FROM AUTHOR]

Morphologically, the lipophagy in yeast cell mimics microautophagy, which includes a direct amendment of the vacuolar membrane that engulfs lipid droplets (LDs). The molecular mechanism of the membrane modifications that elicits microautophagy still remains elusive. In this study, an analysis of membrane lipid distribution at a nanoscale level showed that PtdIns(4)P is localized in the cytoplasmic leaflet of microautophagic vesicles, which are derived when the vacuole's membrane domains engulfed LDs both in the stationary phase and in acute nitrogen starvation. Furthermore, the PtdIns(4)P-positive raft-like domains engulf LDs through a microautophagic mechanism. When single temperature-conditional mutants of STT4 or PIK1 PtdIns 4-kinases were used, in the vacuole of STT4 and PIK1 mutant cells, microautophagic vesicles drastically decreased at restrictive temperatures, and the labeling density of PtdIns(4)P on the microautophagic vesicles and the sizes of the mutants' microautophagic vesicles also decreased. These results suggest that both Stt4p and Pik1p have important roles in the microautophagy of the vacuole in the stationary phase and under nitrogen starvation conditions. Unlabelled Image • PtdIns(4)P is located on the cytoplasmic leaflets of the microautophagic vesicles. • PtdIns(4)P is localized on the vacuolar membrane contacting with lipid droplet. • Both Stt4p and Pik1p have essential roles in the microautophagy of the vacuole. [ABSTRACT FROM AUTHOR]

Autophagy is regulated by phosphoinositides. We have previously shown that phosphatidylinositol 4-phosphate (PtdIns(4)P) is localized in the autophagosomal membrane. Additionally, in yeast cells, phosphatidylinositol 4-kinases Pik1p and Stt4p play important roles in the formation of the autophagosome and its fusion with the vacuole, respectively. In this study, we analyzed the primary role of PtdIns(4)P phosphatases in yeast autophagy. The PtdIns(4)P labeling densities in the membranes of the vacuoles, mitochondria, nucleus, endoplasmic reticulum, and plasma membrane dramatically increased in the phosphatase deletion mutants sac1 ∆ and sjl3 ∆, and the temperature-sensitive mutant sac1 ts/ sjl3 ∆ at the restrictive temperature. GFP-Atg8 processing assay indicated defective autophagy in the sac1 ∆ and sac1 ts/ sjl3 ∆ mutants. In contrast to the localization of PtdIns(4)P in the luminal leaflet of autophagosomal membranes in the wild-type yeast, PtdIns(4)P was localized in both the luminal and cytoplasmic leaflets of the autophagosomal membranes in the sac1 ∆ strain. In addition, the number of autophagic bodies in the vacuole significantly decreased in the sac1 ∆ strain, although autophagosomes were present in the cytoplasm. In the sac1 ts/ sjl3 ∆ strain, the number of autophagosomes in the cytoplasm dramatically decreased at the restrictive temperature. Considering that the numbers of autophagosomes and autophagic bodies in the sjl3 ∆ strain were comparable to those in the wild-type yeast, we found that the autophagosome could not be formed when PtdIns(4)P phosphatase activities of both Sac1p and Sjl3p were diminished. Together, these results indicate that the turnover of PtdIns(4)P by phosphatases is essential for autophagosome biogenesis. [Display omitted] • Distribution of PtdIns(4)P increases with decrease in phosphatase activity. • Decrease in PtdIns(4)P phosphatase activity causes defects in autophagy. • Defects occur in autophagosome formation when Sac1p and Sjl3p activities are diminished. • Sac1p is essential for fusion of autophagosome with vacuole. [ABSTRACT FROM AUTHOR]