6). PtdEtn in ER membranes associated to mitochondria (MAMs), with a parallel increase in PtdSer. Fluorescent-labeled PtdSer (NBD-PtdSer) transport assays exhibited that transport of NBD-PtdSer from your ER to both mitochondria and endosomes and/or vacuole is usually affected inbtn1-cells. Moreover,btn1-affects the synthesis of PtdEtn by the Kennedy pathway and impairs the ability ofpsd1-cells to restore PtdEtn to normal levels in mitochondria and vacuoles by ethanolamine addition. In summary, lack of Btn1p alters phospholipid levels and might play a role in regulating their subcellular distribution. == INTRODUCTION == We have previously reported that theSaccharomyces cerevisiae BTN1gene encodes a nonessential protein that is 39% identical and 59% much like humanCLN3(Pearce and Sherman, 1997). Mutations in humanCLN3, which codes for any lysosomal transmembrane protein, causes Batten disease, the juvenile form of neuronal ceroid lipofuscinosis (NCL) (The International Batten Disease Consortium, 1995;Jarvela et al., 1998;Kyttala et al., 2004;Storch et al., 2004;Phillips et al., 2005). Btn1p is located in the vacuole, the yeast membrane compartment that is equivalent to the mammalian lysosomal organelle (Croopnick et al., 1998;Pearce et al., 1999), although several recent studies in aS. cerevisiaemodel found Btn1p associated with punctate membrane structures (Vitiello et al., 2010;Wolfe et al., 2011). Furthermore, localization studies of overexpressed Btn1p inSchizosaccharomyces pombeassociated this protein to Golgi membranes (Codlin and Mole, 2009). The primary function of this protein remains unknown; however, Btn1p has been implicated in several cellular pathways. In aS. cerevisiaeyeast model, lack of Btn1p (btn1-) led to a decreased vacuolar pH during the logarithmic phase of growth (Pearce et al., 1999). Furthermore,btn1-cells downregulate ATP hydrolysis by vacuolar-type H+-ATPase (V-ATPase) most likely as a cell response to control H+transport across the vacuolar membrane and compensate for pH imbalance caused by the lack of Btn1p (Padilla-Lopez and Pearce, 2006). This alteration at the vacuole has been implicated in deficient regulation of intracellular L-arginine levels, withbtn1-cells showing a lower ability to transport this amino acid into isolated vacuoles in vitro (Kim Edivoxetine HCl et al., 2003). However, this latter defect could be a secondary result from the primary vacuolar pH alteration observed inbtn1-cells. Little is known about the mechanisms regarding lipid transport and distribution within eukaryotic cells. PtdSer is usually synthesized by PtdSer synthase in a specialized region of the endoplasmic reticulum (ER) membrane that is associated to mitochondria [mitochondria-associated membrane (MAM)] (Gaigg et Edivoxetine HCl al., 1995). PtdSer is usually transported to the locus of PtdSer decarboxylases Psd1p Mouse monoclonal to GAPDH or Psd2p, where is usually decarboxylated to form phosphatidylethanolamine (PtdEtn). Psd1p is usually a well-known inner mitochondrial protein (Kuchler et al., 1986). However, whereas previous studies associated Psd2p to Golgi-vacuole membranes (Trotter et al., 1993;Trotter and Voelker, 1995), a recent study refined Psd2p localization to the endosome, arguing that this enzyme controls vacuolar membrane phospholipid content (Gulshan et al., 2010). Following its formation, PtdEtn is transported back to the ER for the synthesis of phosphatidylcholine (PtdCho) (Atkinson et al., 1980;Trotter et al., 1993). Alternatively, PtdEtn can be synthesized through the cytidyldiphosphate (CDP)-ethanolamine branch of the Kennedy pathway (Kennedy and Weiss, 1956). PtdEtn is essential for growth of the yeastS. cerevisiaeand is required for the function and integrity of mitochondrial membranes; a lack of the major PtdSer decarboxylase, Psd1p, prospects to a substantial decrease of PtdEtn in total cellular and mitochondrial membranes, conferring a petite phenotype (Birner et al., 2001). Moreover, depletion of PtdEtn causes defects in the assembly of mitochondrial protein complexes and loss of mitochondrial DNA (Birner et al., 2001;Storey et al., 2001;Birner et al., 2003). PtdEtn also plays a crucial role in GPI anchor biosynthesis (Menon and Stevens, 1992) and seems to be involved in the autophagy and cytosol-to-vacuole-targeting delivery pathways (Huang and Klionsky, 2002;Wang and Klionsky, 2003;Yoshimori, 2004;Nebauer et al., 2007). We statement a potential newly identified role for Btn1p: being necessary for the distribution of phospholipids in cell membranes. We statement that yeast cells lackingBTN1show decreased transport of PtdSer from ER membranes to Edivoxetine HCl either mitochondria or endosome and/or vacuole membranes, resulting in a significant decrease of PtdEtn synthesis in cells lacking Psd1p (psd1-). Furthermore, a lack of Btn1p (btn1-) also partially impairs the synthesis of PtdEtn by the Kennedy pathway. Thus, the multiple cellular phenotypes associated to a lack of Btn1p, or indeed CLN3, could be a result of altered membrane phospholipid content. == RESULTS == == Btn1p is not a component of the V-ATPase complex == Our previous study.
