Membrane Topology of Mouse Stearoyl-CoA Desaturase 1

Abstract

Stearoyl-CoA desaturase (SCD) is an integral membrane protein anchored in the endoplasmic reticulum. It catalyzes the biosynthesis of monounsaturated fatty acids that are required for the synthesis of triglycerides, cholesteryl esters, and phospholipids. Four mouse isoforms of SCD (SCD1-4) and two human isoforms have been characterized. In the current study, we characterize the topology of the mouse SCD1 isoform. Hydropathy analysis of the 355-amino acid mouse SCD1 protein predicts that the protein contains four transmembrane domains (TMDs) and three loops connecting the membrane-spanning domains. To define the topology of the protein, recombinant SCD1 constructs containing epitope tags were transiently expressed in HeLa cells and analyzed by indirect immunofluorescence and cysteine derivatization. Our data provide evidence that the N and C termini of SCD1 are oriented toward the cytosol with four transmembrane domains separated by two very short hydrophilic loops in the ER lumen and one large hydrophilic loop in the cytosol. In addition, based on the previous observation that SCD is a thiol enzyme, we sought to investigate whether the cysteine residues were essential for enzyme activity through mutagenesis studies, and our data suggest that the cysteines in SCD are not catalytically essential. Stearoyl-CoA desaturase (SCD)2 is the rate-limiting enzyme responsible for de novo synthesis of monounsaturated fatty acids from saturated fatty acids (1-4). It is an iron-containing enzyme that catalyzes the insertion of the cis double bond at the ⌬9 position of fatty acyl-CoAs in conjunction with cytochrome b 5 reductase, cytochrome b 5 , and the cofactor NADH (5-7). The preferred substrates of SCD are palmitoyl (C16:0)-CoA and stearoyl (C18:0)-CoA, which are converted into palmitoleoyl (C16:1)-CoA and oleoyl (C18:1)-CoA, respectively (8).A number of mammalian SCD genes have been cloned and studied. Two SCD genes have been cloned in rats (9) and four (SCD1-4) in mice (10 -13). In human, two SCD genes (hSCD1 and hSCD5) have been characterized (14, 15). The mouse isoforms share a high degree of amino acid homology (Ͼ70%), but among them, the mouse SCD1 is the best understood. It is comprised of 355 amino acids and is highly regulated by dietary and hormonal factors, including glucose (16), fructose (17), cholesterol (18), polyunsaturated fatty acids (19,20), insulin (17, 21), and leptin (22). Research in recent years has unveiled the significant role of SCD1 in lipid metabolism. The products of SCD, palmitoleic and oleic acids, are the most abundant fatty acids found in triglycerides and cholesteryl esters. Significant reduction in tissue triglycerides and cholesteryl esters has been observed in a laboratory mouse model with targeted disruption in the SCD1 gene (SCD1Ϫ/Ϫ) (23). It was also found that the SCD1Ϫ/Ϫ mice have very low levels of triglycerides in the very low density lipoprotein fraction compared with the wild-type counterparts (23). More interestingly, SCD1 is the major target gene for leptin, and SCD1 deficien...

Biosynthesis of polyunsaturated fatty acids in lower eukaryotes

“…The same authors suggested that this six-TMH-model could be valid only for acyl-lipid desaturases and not for desaturases that use soluble substrates as acylCoA. This was recently confirmed by the determination of the topology for the mouse stearoyl CoA desaturase 1 (28), which is in good agreement to the classical model (16) consisting of four TMHs (Fig. 1).…”

A Small Membrane-peripheral Region Close to the Active Center Determines Regioselectivity of Membrane-bound Fatty Acid Desaturases from Aspergillus nidulans

“…Our experimental approach was to determine protein domains of the ⌬12/3 bifunctional desaturase, An1, sufficient to install bifunctionality in the frame of the monofunctional ⌬12 desaturase, An2. Based on secondary structure predictions using TMHMM (34) and PredictProtein (35), we identified the sequence stretches of An2 and An1 corresponding to the membrane-spanning and cytosolic domains previously postulated for membrane-bound fatty acid desaturases (7)(8)(9)36). In analogy to previous studies, the sequences of An2 and An1 were divided into the consecutive domains A through I, as follows ( Fig.…”

“…However, the majority of desaturase enzymes reside within membranes, and up to now the principles of membrane-bound desaturase specificity are not understood. Moreover, structural information on membrane-bound desaturases is limited, so only hydropathy and topology analyses are available, predicting the formation of four transmembrane domains and either one or two membrane-peripheral protein domains (7)(8)(9). Three highly conserved histidine-rich motifs are essential for catalysis (1,10), and it was proposed that these histidine clusters coordinate two iron atoms in the active site (11).…”

Role of stearoyl-coenzyme A desaturase in regulating lipid metabolism

“…Scd1 transcription and the half life of the protein are increased in the presence of the PPARα-agonist clofibric acid [36 • ,37]. Mouse SCD1 contains four transmembrane domains with the N and C-termini oriented towards the cytosol [38]. The N-terminus contains a sequence responsible for the rapid degradation of SCD1 in a mechanism that involves the ubiquitinproteasome-dependent system [34,35].…”