Differential reliance of CTD-nuclear envelope phosphatase 1 on its regulatory subunit in ER lipid synthesis and storage.

Lipin 1 is an ER enzyme that produces diacylglycerol, the lipid intermediate that feeds into the synthesis of glycerophospholipids for membrane expansion or triacylglycerol for storage into lipid droplets. CTD-Nuclear Envelope Phosphatase 1 (CTDNEP1) regulates lipin 1 to restrict ER membrane synthesis, but a role for CTDNEP1 in lipid storage in mammalian cells is not known. Furthermore, how NEP1R1, the regulatory subunit of CTDNEP1, contributes to these functions in mammalian cells is not fully understood. Here, we show that CTDNEP1 is reliant on NEP1R1 for its stability and function in limiting ER expansion. CTDNEP1 contains an amphipathic helix at its N-terminus that targets to the ER, nuclear envelope and lipid droplets. We identify key residues at the binding interface of CTDNEP1 and NEP1R1 and show that they facilitate complex formation in vivo and in vitro. We demonstrate that NEP1R1 binding to CTDNEP1 shields CTDNEP1 from proteasomal degradation to regulate lipin 1 and restrict ER size. Unexpectedly, NEP1R1 was not required for CTDNEP1's role in restricting lipid droplet biogenesis. Thus, the reliance of CTDNEP1 function on NEP1R1 depends on cellular demands for membrane production versus lipid storage. Together, our work provides a framework into understanding how the ER regulates lipid synthesis under different metabolic conditions.

Differential reliance of CTD-nuclear envelope phosphatase 1 on its regulatory subunit in ER lipid synthesis and storage.

The endoplasmic reticulum (ER) is the site for the synthesis of the major membrane and storage lipids. Lipin 1 produces diacylglycerol, the lipid intermediate critical for the synthesis of both membrane and storage lipids in the ER. CTD-Nuclear Envelope Phosphatase 1 (CTDNEP1) regulates lipin 1 to restrict ER membrane synthesis, but its role in lipid storage in mammalian cells is unknown. Here, we show that the ubiquitin-proteasome degradation pathway controls the levels of ER/nuclear envelope-associated CTDNEP1 to regulate ER membrane synthesis through lipin 1. The N-terminus of CTDNEP1 is an amphipathic helix that targets to the ER, nuclear envelope and lipid droplets. We identify key residues at the binding interface of CTDNEP1 with its regulatory subunit NEP1R1 and show that they facilitate complex formation in vivo and in vitro . We demonstrate a role for NEP1R1 in temporarily shielding CTDNEP1 from proteasomal degradation to regulate lipin 1 and restrict ER size. Unexpectedly, we found that NEP1R1 is not required for CTDNEP1's role in restricting lipid droplet biogenesis. Thus, the reliance of CTDNEP1 function on its regulatory subunit differs during ER membrane synthesis and lipid storage. Together, our work provides a framework into understanding how the ER regulates lipid synthesis and storage under fluctuating conditions.