Calcium-dependent and -independent lipid transfer mediated by tricalbins in yeast

Membrane contact sites (MCSs) formed between the endoplasmic reticulum (ER) and the plasma membrane (PM) provide a platform for nonvesicular lipid exchange. The ER-anchored tricalbins (Tcb1, Tcb2, and Tcb3) are critical tethering factors at ER-PM MCSs in yeast. Tricalbins possess a synaptotagmin-like mitochondrial-lipid-binding protein (SMP) domain and multiple Ca²⁺-binding C2 domains. Although tricalbins have been suggested to be involved in lipid exchange at the ER-PM MCSs, it remains unclear whether they directly mediate lipid transport. Here, using in vitro lipid transfer assays, we discovered that tricalbins are capable of transferring phospholipids between membranes. Unexpectedly, while its lipid transfer activity was markedly elevated by Ca²⁺, Tcb3 constitutively transferred lipids even in the absence of Ca²⁺. The stimulatory activity of Ca²⁺ on Tcb3 required intact Ca²⁺-binding sites on both the C2C and C2D domains of Tcb3, while Ca²⁺-independent lipid transport was mediated by the SMP domain that transferred lipids via direct interactions with phosphatidylserine and other negatively charged lipid molecules. These findings establish tricalbins as lipid transfer proteins, and reveal Ca²⁺-dependent and -independent lipid transfer activities mediated by these tricalbins, providing new insights into their mechanism in maintaining PM integrity at ER-PM MCSs.