Panels a and b show cells with cellular blebbing (black arrows), visible nuclei (labeled N) and several vacuoles (black arrowheads) together with lysosomal proliferation (white arrowheads). AT-1 is essential for cell viability as its downregulation results in widespread cell death and induction of features characteristic of autophagy. and (Hirabayashi et al., 2004), and is upregulated as result of ER-induced stress, suggesting a possible role during the unfolded protein response (UPR) (Shaffer et al., 2004). Recent work has also shown that is upregulated in motor neurons of 7-Epi-docetaxel patients affected by sporadic amyotrophic lateral sclerosis (ALS) (Jiang et al., 2007) and mutated in patients affected by autosomal dominant spastic paraplegia-42 (SPG42) (Lin et al., 2008), suggesting an implication in neurodegenerative disorders. Here, we report that AT-1 (also called solute carrier family 33 member 1, SLC33A1) is an ER membrane acetyl-CoA transporter. AT-1 regulates the acetylation of BACE1, LDLR, APP and other ER-based protein substrates, and is upregulated in the brain of late-onset (sporadic) AD patients. Importantly, we show that AT-1 is essential 7-Epi-docetaxel for cell viability because its downregulation results in widespread cell death and induction of features characteristic of autophagy. These studies point to a fundamental role of the ER-based acetylation machinery in both physiological and pathological conditions. Results AT-1 is the ER membrane acetyl-CoA transporter To assess whether AT-1 is responsible for the acetyl-CoA transport activity that we have identified and described in the ER membrane (Costantini et al., 2007), and to characterize its biochemical properties 7-Epi-docetaxel together with its disease-relevant functions, we generated several individual colonies of Chinese hamster ovary (CHO) cells that overexpress human AT-1 (Fig. 1A,B). Transgenic AT-1 displayed ER localization and was completely absent from Golgi fractions (Fig. 1C), which is usually consistent with our previous localization of the ER membrane acetyl-CoA transport activity (Costantini et al., 2007). Next, we assayed the acetyl-CoA transport activity in individual fractions from a subcellular fractionation gradient of control (non-transfected) cells. The assay was performed under native conditions and in the absence of detergents, Rabbit Polyclonal to SCFD1 which preserves biochemical properties as well as in vivo topographical orientation of the membranes (Carey and Hirschberg, 1981; Costantini et al., 2007; Ko and Puglielli, 2009; Puglielli et al., 1999a; Puglielli et al., 1999b). Fig. 1D shows that the acetyl-CoA transport activity was only observed in fractions corresponding to the ER and that the distribution pattern of AT-1 overlapped with the endogenous acetyl-CoA membrane transport activity. Additionally, when we compared the acetyl-CoA membrane transport activity of comparable ER fractions generated from control and AT-1 overexpressing cells, we found a significant increase in the rate of acetyl-CoA translocation following AT-1 overexpression (Fig. 1E). Open in a separate windows Fig. 1. AT-1 localizes in the ER and stimulates acetyl-CoA transport across the ER membrane. (A,B) Western blot analysis shows successful transfection of AT-1 into CHO cells. AT-1 migrates very close to the predicted molecular mass of 61 kDa. Transgenic AT-1 contained a Myc-His tag at the C-terminus and could be acknowledged with 7-Epi-docetaxel both anti-AT-1 (A) and anti-Myc (B) antibodies. (C) The subcellular distribution of transgenic AT-1 was analyzed by SDS-PAGE and immunoblotting after separation of intracellular membranes on a 10C24% discontinuous Nycodenz gradient. The appropriate subcellular markers are indicated. (D) Intracellular membranes from control CHO cells were prepared as described in C and then assayed for acetyl-CoA transport activity. Results are the average + s.d. (mRNA were already evident 2 days after siRNA treatment (Fig. 3A), strong changes in the protein levels could only be observed after 6 days of treatment, suggesting a long half-life of the transporter (Fig. 3B). Surprisingly, the downregulation of AT-1 was accompanied by widespread cell death as assessed by conventional field microscopy (Fig. 3C), calcein incorporation into living cells (Fig. 3C), and direct counting of cells stained with a live-dead assay (Fig. 3D,E). Such an effect was not observed when cultures were treated with non-silencing siRNA (Fig. 3CCE) or with other non-related siRNAs previously used (Costantini et al., 2006; Jonas et al., 2008; Ko and Puglielli, 2007). These results should be viewed together with the fact that downregulation of AT-1 in zebrafish.