CHP1 promotes lipid droplet growth and regulates the localization of key enzymes for triacylglycerol synthesis

The glycerol-3-phosphate (G-3-P) pathway is central to the synthesis of triacylglycerols (TAGs) and glycerophospholipids, essential for membrane biogenesis and lipid storage. The first and rate-limiting step in this pathway is catalyzed by glycerol-3-phosphate acyltransferases (GPATs), with microsomal GPAT3 and GPAT4 being evolutionarily conserved and predominant in most tissues. While previous studies have implicated Calcineurin B homologous protein 1 (CHP1) as a cofactor for GPAT4, the broader role of CHP1 in regulating microsomal GPATs and TAG biosynthesis remains unclear. Here, we demonstrate that CHP1 is a critical regulator of both GPAT3 and GPAT4, essential for their stability, enzymatic activity, and lipid droplet (LD) localization. Structural modeling and mutational analyses identified key hydrophobic interfaces mediating the CHP1-GPAT interaction, which are required for optimal GPAT activity and LD growth. Loss of CHP1 impairs LD expansion and disrupts the localization of GPAT3/4 and downstream enzymes in the TAG synthesis pathway, including 1-acylglycerol-3-phosphate O-acyltransferase 3 (AGPAT3) and diacylglycerol O-acyltransferase 2 (DGAT2). Mechanistically, CHP1 helps circumvent seipin-mediated restriction of late LD-targeting enzymes, facilitating their access to mature LDs. Together, our findings reveal CHP1 as a dual-function regulator that stabilizes and activates microsomal GPATs while enabling the coordinated recruitment of TAG biosynthetic enzymes to LDs. This work uncovers a previously unrecognized mechanism for regulating LD growth and glycerolipid metabolism, with broad implications for lipid homeostasis and metabolic diseases.