Membrane topology of mouse stearoyl-CoA desaturase 1.
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.
Man
Weng Chi
WC
Biochemistry andNutritional Sciences, University of Wisconsin-Madison, 53706, USA.
Miyazaki
Makoto
M
Chu
Kiki
K
Ntambi
James M
JM
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry; D000818:Animals; D002384:Catalysis; D002462:Cell Membrane / Q000378:metabolism; D003545:Cysteine / Q000737:chemistry; D017931:DNA Primers / Q000737:chemistry; D019286:Endopeptidase K / Q000737:chemistry; D004721:Endoplasmic Reticulum / Q000378:metabolism; D000939:Epitopes / Q000737:chemistry; D019084:Fluorescent Antibody Technique, Indirect; D006367:HeLa Cells; D006801:Humans; D015151:Immunoblotting; D051379:Mice; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D009154:Mutation; D017830:Octoxynol / Q000494:pharmacology; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D020033:Protein Isoforms; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D012694:Serine / Q000737:chemistry; D013230:Stearoyl-CoA Desaturase / Q000737:chemistry* / Q000378:metabolism
D000596:Amino Acids; D017931:DNA Primers; D000939:Epitopes; D020033:Protein Isoforms; D011994:Recombinant Proteins; D012694:Serine; D017830:Octoxynol; D013230:Stearoyl-CoA Desaturase; D019286:Endopeptidase K; D003545:Cysteine