Evidence That the Yeast Desaturase Ole1p Exists as a Dimer in Vivo

Abstract

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 ⌬ 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 ϳ75% 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.Unsaturated fatty acids are found in the membranes of prokaryotes and eukaryotes. The balance between saturated and unsaturated fatty acids is a key variable in maintaining membrane fluidity. Postsynthetic desaturation of fatty acids is mediated by desaturase enzymes (1-5). Fatty acid desaturases are derived from two evolutionary lineages, the soluble acyl-ACP desaturases found primarily in the plastids of higher plants (e.g. the castor ⌬ 9 -18:0-acyl 2 carrier protein (ACP) 3 desaturase (6) and the integral membrane desaturases typified by the yeast Ole1 ⌬ 9 -desaturase (Ole1p) (7)). The reactions require iron cofactors, molecular oxygen, and reducing equivalents and are both inhibited by azide and cyanide but are insensitive to carbon monoxide. All desaturases investigated to date show remarkable stereo-selectivity in abstracting pro(R) hydrogens from adjacent carbons in the fatty acid substrate (8). As noted by Bloch (1), desaturase selectivity "would seem to approach the limits of the discriminatory power of enzymes" by a mechanism(s) that remain to be determined almost 40 years later.Ole1p is a bifunctional 53-kDa protein (7), composed of two distinct domains: an N-terminal 42-kDa desaturase domain that has 36% homology to the rat stearoyl-CoA desaturase and a C-terminal 11-kDa domain that has strong homology to cytochromes b 5 (9).Although integral membrane desaturases, like many membrane proteins, have proved difficult overexpress and purify, sequence analysis has provided important information regarding the structure of the enzyme. For example, Martin and coworkers (7) employed hydropathy analysis to predict t...

FAD2 and FAD3 Desaturases Form Heterodimers That Facilitate Metabolic Channeling in Vivo

“…5A). (18), in a similar fashion to those reported for soluble desaturases (14). In the present work, we show that plant FAD enzymes form self-and specific heteroassociations when expressed both in yeast and in Arabidopsis protoplasts.…”

“…After stripping the lower epidermis, the leaf was incubated in an enzyme solution containing 1% cellulose Onozuka R10 (Yakult, Tokyo, Japan), 0.25% macerozyme Onozuka R10 (Yakult), 0. (18). Briefly, ϳ5 ϫ 10 4 protoplasts in 0.2 ml of MMg solution were mixed with 30 g of plasmid DNA and diluted 1:1 (v:v) with a freshly prepared solution containing 40% (w/v) PEG (molecular weight, 4000; Fluka-Sigma-Aldrich), 0.1 M CaCl 2 , and 0.2 M mannitol.…”

“…Yeast cells (YPH499) were cultured overnight in SD ϪUra containing 2% glucose at 30°C (18). Upon reaching an A 600 of 0.5, cell pellets were collected by centrifugation and washed with SD ϪUra lacking dextrose, after which the cells were induced at 30°C for 4 h in SD ϪUra supplemented with 2% galactose, 0.5% Tergitol Nonidet P-40, 0.1 mM oleic acid, and 0.1 mM linoleic acid, followed by incubation at 18°C for between 1 and 4 days.…”

“…Because FAD2 and FAD3 can form both homodimers and heterodimers in yeast, we hypothesized that the FAD2-FAD3 heterodimers could form the basis for a metabolic channel capable of converting PC-18:1 to PC-18:3 without free exchange of a PC-18:2 intermediate. We tested this hypothesis by coexpressing FAD2 and FAD3 in yeast and culturing the cells in a medium contain- ing mass labeled oleic acid (in which two hydrogens were replaced by deuterons) or linoleic acid in which the C1 carbon was replaced by 13 C. GAL1 and GAL10 are distinct galactose-inducible promoters that drive luciferase expression at approximately equal levels (18). However, because FAD3 is regulated extensively at the translational and post-transcriptional levels (27,28), we first characterized the relative accumulation of FAD2 and FAD3 polypeptides.…”

“…In a previous study, we presented evidence that functional yeast ⌬ 9 -desaturase Ole1 forms a dimer in vivo (18). To determine whether the observed dimeric organization of the yeast desaturase is shared with other desaturases, we undertook the present study of a series of higher plant membrane desaturases, FAD2, FAD3, FAD6, FAD7, and FAD8, using two independent bimolecular complementation assays.…”