After centrifugation at 15,000 rpm for 10 min, radioactivities in the supernatants were determined as described above
After centrifugation at 15,000 rpm for 10 min, radioactivities in the supernatants were determined as described above. sterol levels (3). Intermembrane sterol transport is considered to be carried out by vesicular and nonvesicular mechanisms. Nonvesicular sterol transport (4,C7) has been proposed to be mediated by lipid transfer proteins and/or through membrane contact sites; however, the mechanism remains elusive (8). Oxysterol-binding protein (OSBP)-related proteins (ORPs), including OSBP homologs (Osh1COsh7) of (10,C12). In contrast, yeast Osh6 and Osh7 and mammalian ORP5 and ORP8 have been reported to transport phosphatidylserine (PS) from the ER to plasma membrane (13, 14). In addition, roles of ORPs as lipid sensors or regulatory proteins in various cellular processes have also been proposed (9). Therefore, the precise molecular functions of ORPs are still debated. In Fucoxanthin genes are essential for cell viability; however, their combined deletion cause lethality, indicating a shared and overlapping essential function (15), which is yet undetermined. Sterols play important roles in the biogenesis and maintenance of mitochondrial membranes (16, 17), and the synthesis of ergosterol, a yeast major sterol, is crucial for mitochondrial morphogenesis in (18). In mammals, cholesterol is transported to mitochondria and used for the synthesis of steroid hormones, oxysterols, and hepatic bile acids (17, 19). However, the molecular mechanisms underlying sterol transport from the ER to mitochondria remain unclear. In this study, we constructed a system to evaluate sterol transport from the ER to mitochondria using membrane fractions from yeast and analyzed the molecular mechanism. Our results suggest that Osh proteins mediate sterol transport from the ER to mitochondria. Results In vitro sterol transport from the ER to mitochondria We have previously constructed a system to analyze sterol transport from the ER to mitochondria in (20). In this system, sterol transport was evaluated by measuring the content of steryl ester synthesized by a mitochondrially targeted recombinant sterol acyltransferase, SatA, from (mito-SatA-EGFP) in a yeast mutant deleted for and encoding acyl-CoA:sterol acyltransferases (using isolated yeast organelle membranes (Fig. 1). In this system, 3H-labeled sterol was first synthesized in the donor ER membrane prepared from the sterol transport reaction. A significant amount of steryl ester was detected in the mitochondrial fraction of the sterol transport with the ER and mitochondrial fractions from the WT strain. sterol transport. represent S.E. sterol transport. Sterol transport was measured using the fractions of the represent S.E. sterol transport. Sterol transport was measured using the fractions of the represent S.E. sterol transport reaction. After the sterol transport reaction, samples were treated with proteinase K in the absence or presence of 0.2% Triton X-100. Cox2 was detected by Western blotting using antibody against Cox2. and strain expressing Osh4-FLAG. The strains expressing Osh4-FLAG were cultured on SD medium at 25 or 37 C. represent S.E. To exclude the effect of Osh proteins associated with membranes, sterol transport was examined using membrane and cytosolic fractions prepared from the strains having genetic backgrounds that bear the wildtype (WT) or the temperature-sensitive allele along with the Fucoxanthin deletions of the other six genes (and using the ER fractions at permissive temperature (25 C). Similar amounts of 3H-labeled sterol were obtained in the ER fractions from both strains (Fig. 4strain compared with that using the fractions from the strain. Next, the effect of Osh4 inactivation was examined using the cytosolic fraction of the WT, strain with the ER and mitochondrial fractions of the strain. The ER with cytosolic fractions and the mitochondrial fraction were separately preincubated at 25 or 37 C for 30 min after which these fractions were mixed and incubated for 1 h (Fig. 4or strain compared with that in the presence of the cytosolic fraction of the WT strain. At 37 C, sterol esterification Rabbit Polyclonal to LMTK3 was similarly lower in the presence of the cytosolic fraction than in the presence of the WT cytosolic fraction. In addition, the sterol esterification was significantly lower in the reaction containing the cytosolic fraction than that with the cytosolic fraction. These results suggest the involvement of genes in the transport of sterol strain at 37 C. The amount of Osh4 added Fucoxanthin to the reaction was calculated according to the reported numbers of Osh4 molecules per cell (27). Although the effect of addition of an equivalent amount of His6-Osh4 to the cytosolic.