Jiangsu Key Laboratory for Molecular and Medical Biotechnology, College of Life Sciences, Nanjing Normal University, Nanjing, China.
Department of Molecular, Cellular and Developmental Biology, University of Colorado, Boulder, Colorado, USA.
J Biol Chem. 2021 Jan-Jun;296:100729. doi: 10.1016/j.jbc.2021.100729. Epub 2021 Apr 29.
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 Ca-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 Ca, Tcb3 constitutively transferred lipids even in the absence of Ca. The stimulatory activity of Ca on Tcb3 required intact Ca-binding sites on both the C2C and C2D domains of Tcb3, while Ca-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 Ca-dependent and -independent lipid transfer activities mediated by these tricalbins, providing new insights into their mechanism in maintaining PM integrity at ER-PM MCSs.
内质网（ER）和质膜（PM）之间形成的膜接触位点（MCSs）为非囊泡脂质交换提供了一个平台。在酵母中，ER 锚定的三钙结合蛋白（Tcb1、Tcb2 和 Tcb3）是 ER-PM MCSs 的关键连接因子。三钙结合蛋白具有突触融合蛋白样线粒体脂质结合蛋白（SMP）结构域和多个 Ca 结合 C2 结构域。尽管三钙结合蛋白被认为参与 ER-PM MCSs 中的脂质交换，但它们是否直接介导脂质运输仍不清楚。在这里，我们使用体外脂质转移测定发现，三钙结合蛋白能够在膜之间转移磷脂。出乎意料的是，尽管其脂质转移活性被 Ca 显著增强，但 Tcb3 即使在没有 Ca 的情况下也会持续地转移脂质。Ca 对 Tcb3 的刺激活性需要 Tcb3 的 C2C 和 C2D 结构域上完整的 Ca 结合位点，而 Ca 独立的脂质转运则由 SMP 结构域介导，该结构域通过与磷脂酰丝氨酸和其他带负电荷的脂质分子的直接相互作用来转移脂质。这些发现确立了三钙结合蛋白作为脂质转移蛋白，并揭示了这些三钙结合蛋白介导的 Ca 依赖性和非依赖性脂质转移活性，为它们在 ER-PM MCSs 中维持 PM 完整性的机制提供了新的见解。
