Differential lipid selectivity of StARD phospholipid transporters revealed by native MS

Intracellular lipid transport in eukaryotes is largely mediated by lipid transfer proteins (LTPs). Transport kinetics differ markedly among lipid species, implying selective lipid recognition by the involved proteins. Here, we characterize endogenous ligands of the human phospholipid transporters STARD2, STARD7 and STARD10 by multistage native mass spectrometry (MS). Our results demonstrate that they exhibit distinct lipid selectivities, with STARD7 binding a broad range of phospholipids, whereas STARD2 and STARD10 preferentially copurify with poly- and di-unsaturated phospholipids, respectively. We link this acyl chain selectivity to tissue-specific LTP expression patterns and show that LTP expression levels modulate lipid biosynthesis. Through site-directed mutagenesis and molecular dynamics simulations, we further identify a conserved arginine that is essential for phospholipid binding in STARD7 but dispensable in STARD2 and STARD10. To investigate regulation of LTP activity, we mapped phosphorylation sites by native top-down MS and found that STARD2 and STARD10 are phosphorylated in membrane-binding regions. Liposome-based assays revealed that phosphorylation abolishes lipid transfer activity of STARD10 and that lipid selectivity influences the transfer rates of different lipid probes. Together, our results demonstrate that LTPs exhibit distinct lipid binding preferences and suggest that cells finely tune lipid homeostasis through regulating LTP expression levels and activity.