Identification of Cellular Sensors for Unsaturated Fatty Acids

In mammalian cells fatty acids (FAs) are required for the synthesis of membrane phospholipid components and energy generation. However, overaccumulation of FA is toxic. Accumulation of FAs prevents their further synthesis by stabilization of Insulin-induced gene 1(Insig-1), an ER membrane protein that inhibits proteolytic activation of sterol regulatory element binding protein-1 (SREBP-1), a transcription factor that activates all genes required for FA synthesis. This regulatory reaction is stimulated by unsaturated but not saturated FAs. Unsaturated FAs stabilize Insig-1 through disrupting the interaction between Insig-1 and UBX domain-containing protein 8 (Ubxd8), which recruits p97, a protein required for degradation of endoplasmic-reticulum (ER) membrane proteins, to Insig-1. Here, we identified Ubxd8, a protein that does not contain any previously recognized domains that bind FAs, as a sensor for unsaturated FAs. In cultured cells, we demonstrated that unsaturated FAs but not saturated FAs stimulated polymerization of Ubxd8 through bimolecular fluorescence complementation (BiFC) assays. In vitro, unsaturated but not saturated FAs also stimulated polymerization of purified recombinant Ubxd8. The effect of different FAs and their derivatives on polymerization of Ubxd8 in vitro correlated with their effect on stabilization of Insig-1 in cultured cells. Ubxd8 contains 3 domains, namely ubiquitin X (UBX), ubiquitin associated (UBA) and ubiquitin associating (UAS) domain. Deletion analysis showed that UAS domain was necessary and sufficient for polymerization of the protein in response to unsaturated FAs. Point mutations in UAS domain that disrupted its interaction with unsaturated FAs in vitro also impaired the ability of full length Ubxd8 to stabilize Insig-1 in response to unsaturated FAs in cultured cells. In addition to Ubxd8, the only other protein expressed in mammalian cells that contains a UAS domain similar to that of Ubxd8 is Fas-associated factor 1 (FAF1). We showed that unsaturated FAs also specifically induced polymerization of FAF1, and this polymerization was mediated by the UAS domain. The identification of UAS domain as a motif polymerizing upon interaction with unsaturated FAs should provide more insights into cellular responses to FAs.