Hence, nutrient conditions influence the biosynthesis of M(IP)2C

Hence, nutrient conditions influence the biosynthesis of M(IP)2C in yeast. Autophagy is a catabolic membrane-trafficking phenomenon that occurs in response to drastic changes in the nutrients available to yeast cells, for example during starvation for nitrogen (N) or carbon (Abeliovich & Klionsky, 2001). Although both autophagy and the M(IP)2C content of yeast membranes seem to be responsive to nutritional stress, a direct link between these processes has not been investigated in yeast to date. http://www.selleckchem.com/products/sch772984.html Hence, the question arises as to whether Δipt1 or Δskn1 single and double deletion mutants

are characterized by an altered autophagic response as compared with the corresponding wild type (WT). Therefore, in this study, we used N starvation to assess differences in the autophagic response of the different Δipt1 and/or Δskn1 deletion mutants as compared with WT, as well as their sphingolipid profiles and putative induction of apoptosis, which has previously been linked to autophagy (Maiuri et al., 2007; Scott

et al., 2007). Because overexpression of autophagy-related protein 1, Atg1, in Drosophila was previously shown to induce autophagy and to cause cell death accompanied by increased DNA fragmentation (Scott et al., 2007), we further assessed DNA fragmentation upon N starvation in all mutants and WT. The yeast strains used are Saccharomyces cerevisiae BY4741 (MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0) and the corresponding Δipt1, Δskn1 (Invitrogen, Carlsbad, selleck chemicals llc CA) mutants and the double Δipt1Δskn1 deletion mutant (Thevissen et al., 2005), the pho8Δ60∷pho8 pho13Δ∷kan-lox Flavopiridol (Alvocidib) strain (WT, YTS158) (Noda et al., 1995) and the corresponding Δatg1, Δipt1, Δskn1 and Δipt1Δskn1 mutants. Overnight cultures in YPD medium (1% yeast extract; 2% peptone, 2% glucose) were transferred to SD medium [0.8 g L−1 complete amino acid supplement mixture (Bio 101 Systems); 6.5 g L−1 yeast nitrogen base (YNB); 20 g L−1 glucose] at a start OD600 nm=0.2, grown to the exponential phase till

OD600 nm=0.8, washed twice with SD-N medium (0.17% YNB w/o ammonium sulfate and amino acids, 2% glucose) and shifted to SD-N medium for 4 h. As a control, cells were also shifted to SD medium after reaching the exponential phase. For monitoring bulk autophagy, the alkaline phosphatase activity of Pho8Δ60 was carried out as described previously (Noda et al., 1995; Klionsky, 2007). The percentage of autophagy of the different mutants was relative to the WT autophagy level under the different conditions. After challenge with SD-N medium, cell numbers were measured (using CASY cell counter), ROS levels were determined (via staining with dihydroethidium) and phosphatidylserine externalization of the yeast cultures (via staining with fluorescein isothiocyanate-labeled annexin V in combination with propidium iodide) was quantified using flow cytometry and bd facsdiva software (Madeo et al., 1997; Büttner et al.

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