Evidence that the yeast desaturase Ole1p exists as a dimer in vivo.
Desaturase enzymes are composed of two classes, the structurally well characterized soluble class found predominantly in the plastids of higher plants and the more widely distributed but poorly structurally defined integral membrane class. Despite their distinct evolutionary origins, the two classes both require an iron cofactor and molecular oxygen for activity and are inhibited by azide and cyanide, suggesting strong mechanistic similarities. The fact that the soluble desaturase is active as a homodimer prompted us test the hypothesis that an archetypal integral membrane desaturase from Saccharomyces cerevisiae, the Delta(9)-acyl-Co-A desaturase Ole1p, also exhibits a dimeric organization. Ole1p was chosen because it is one of the best characterized integral membrane desaturase and because it retains activity when fused with epitope tags. FLAG-Ole1p was detected by Western blotting of immunoprecipitates in which anti-Myc antibodies were used for capture from yeast extracts co-expressing Ole1p-Myc and Ole1p-FLAG. Interaction was confirmed by two independent bimolecular complementation assays (i.e. the split ubiquitin system and the split luciferase system). Co-expression of active and inactive Ole1p subunits resulted in an approximately 75% suppression of the accumulation of palmitoleic acid, demonstrating that the physiologically active form of Ole1p in vivo is the dimer in which both protomers must be functional.
Lou
Ying
Y
Biology Department, Brookhaven National Laboratory, Upton, New York 11973, USA.
Shanklin
John
J
D000595:Amino Acid Sequence; D002462:Cell Membrane / Q000378:metabolism; D019281:Dimerization; D000939:Epitopes / Q000737:chemistry; D044943:Fatty Acid Desaturases / Q000737:chemistry* / Q000378:metabolism*; D005227:Fatty Acids / Q000737:chemistry; D005816:Genetic Complementation Test; D006639:Histidine / Q000737:chemistry; D007501:Iron / Q000737:chemistry; D008055:Lipids / Q000737:chemistry; D008957:Models, Genetic; D008969:Molecular Sequence Data; D010100:Oxygen / Q000737:chemistry; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000378:metabolism; D013230:Stearoyl-CoA Desaturase
D000939:Epitopes; D005227:Fatty Acids; D008055:Lipids; D006639:Histidine; D007501:Iron; D044943:Fatty Acid Desaturases; D013230:Stearoyl-CoA Desaturase; C029627:delta-9 fatty acid desaturase; D010100:Oxygen
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