Evidence that diacylglycerol acyltransferase 1 (DGAT1) has dual membrane topology in the endoplasmic reticulum of HepG2 cells.
ABSTRACT Triacylglycerol (TAG) synthesis and secretion are important functions of the liver that have major impacts on health, as overaccumulation of TAG within the liver (steatosis) or hypersecretion of TAG within very low density lipoproteins (VLDL) both have deleterious metabolic consequences. Two diacylglycerol acyltransferases (DGATs 1 and 2) can catalyze the final step in the synthesis of TAG from diacylglycerol, which has been suggested to play an important role in the transfer of the glyceride moiety across the endoplasmic reticular membrane for (re)synthesis of TAG on the lumenal aspect of the endoplasmic reticular (ER) membrane (Owen, M., Corstorphine, C. C., and Zammit, V. A. (1997) Biochem. J. 323, 17-21). Recent topographical studies suggested that the oligomeric enzyme DGAT1 is exclusively lumen facing (latent) in the ER membrane. By contrast, in the present study, using two specific inhibitors of human DGAT1, we present evidence that DGAT1 has a dual topology within the ER of HepG2 cells, with approximately equal DGAT1 activities exposed on the cytosolic and lumenal aspects of the ER membrane. This was confirmed by the observation of the loss of both overt (partial) and latent (total) DGAT activity in microsomes prepared from livers of Dgat1(-/-) mice. Conformational differences between DGAT1 molecules having the different topologies were indicated by the markedly disparate sensitivities of the overt DGAT1 to one of the inhibitors. These data suggest that DGAT1 belongs to the family of oligomeric membrane proteins that adopt a dual membrane topology.
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ABSTRACT: CD38 catalyzes the synthesis of two structurally distinct messengers for Ca2 +-mobilization, cyclic ADP-ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP), from cytosolic substrates, NAD and NADP, respectively. CD38 is generally thought of as a type II membrane protein with its catalytic site facing outside. We recently showed that CD38 exists, instead, in two opposite membrane orientations. The determinant for the membrane topology is unknown. Here, specific antibodies against type III CD38 were designed and produced. We show that mutating the positively charged residues in the N-terminal tail of CD38 converted its orientation to type III, with the catalytic domain facing the cytosol and it was fully active in producing intracellular cADPR. Changing the serine residues to aspartate, which is functionally equivalent to phosphorylation, had a similar effect. The mutated CD38 was expressed intracellularly and was un-glycosylated. The membrane topology could also be modulated by changing the highly conserved di-cysteine. The results indicate that the net charge of the N-terminal segment is important in determining the membrane topology of CD38 and that the type III orientation can be a functional form of CD38 for Ca2 +-signaling. This article is part of a Special Issue entitled: 13th European Symposium on Calcium.Biochimica et Biophysica Acta (BBA) - Molecular Cell Research 11/2014; DOI:10.1016/j.bbamcr.2014.10.028 · 5.30 Impact Factor
Article: The life cycle of lipid droplets[Show abstract] [Hide abstract]
ABSTRACT: Proteomic studies have revealed many potential functions of cytoplasmic lipid droplets, and recent activity has confirmed that these bona fide organelles are central not only for lipid storage and metabolism, but for development, immunity, and pathogenesis by several microbes. There has been a burst of recent activity on the assembly, maintenance and turnover of lipid droplets that reveals fresh insights. This review summarizes several novel findings in initiation of lipid droplet assembly, protein targeting, droplet fusion, and turnover of droplets through lipophagy. Copyright © 2015 Elsevier Ltd. All rights reserved.Current Opinion in Cell Biology 02/2015; 33C:119-124. DOI:10.1016/j.ceb.2015.02.002 · 8.74 Impact Factor
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ABSTRACT: Diacylglycerol acyltransferase-1 (DGAT1) is involved in the assembly of hepatitis C virus (HCV) by facilitating the trafficking of the HCV core protein to the lipid droplet. Here, we abrogated DGAT1 expression in Huh-7.5 cells using either the transcription activator-like effector nuclease (TALEN) or shRNA-lentivirus, and achieved complete, long-term silencing of DGAT1. HCV entry was severely impaired in DGAT1-silenced Huh-7.5 cell lines, which showed markedly diminished claudin-1 (CLDN1) expression. In DGAT1-silenced cell lines, the forced expression of CLDN1 restored HCV entry, implying that the downregulation of CLDN1 is a critical factor underlying defective HCV entry. The expression of hepatocyte nuclear factor 4α (HNF4α) and other hepatocyte-specific genes was also reduced in DGAT1-silenced cell lines. After DGAT1 gene rescue, CLDN1 expression was preserved, and HCV entry was restored. Strikingly, after DGAT1 silencing, CLDN1 expression and HCV entry were also restored by low dose palmitic acid treatment, indicating that the downregulation of CLDN1 was associated with altered fatty acid homeostasis in the absence of DGAT1. Our findings provide novel insight into the role of DGAT1 in the life cycle of HCV.Journal of Virology 06/2014; 88(16). DOI:10.1128/JVI.01428-14 · 4.65 Impact Factor