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STAR Protocols · 2022 · IF 0

In vitro and in vivo assay of the ER lipid scramblase TMEM41B

Lingzhi Wu,Lu Liu,Bolin Xu,Dong Huang,Xiao-Wei Chen

Summary Amphipathic phospholipids translocated by scramblases play a central role in facilitating lipid movement across the membrane bilayer, especially at the endoplasmic reticulum (ER) membranes. Here, we present a protocol for assessing the activity of the ER-localized lipid scramblase TMEM41B. We detail an in vitro fluorescent liposome-based phospholipid scrambling assay and in vivo metabolic labeling in living cells using alkyne-choline. The scramblase activity of other VTT (VMP1, TMEM41, and Tvp38) domain-containing proteins, such as TMEM41A and VMP1, can be assayed. For complete details on the use and execution of this protocol, please refer to Huang et al. (2021) .

研究领域: 细胞生物学 · 分子生物学 · 生物化学

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JOURNAL OF BIOCHEMISTRY · 2023 · IF 2.7

A lipid scramblase TMEM41B is involved in the processing and transport of GPI-anchored proteins

Cao Shu-Ya,Liu Yi-Shi,Gao Xiao-Dong,Kinoshita Taroh,Fujita Morihisa

Protein modification by glycosylphosphatidylinositol (GPI) takes place in the endoplasmic reticulum (ER). GPI-anchored proteins (GPI-APs) formed in the ER are transported to the cell surface through the Golgi apparatus. During transport, the GPI-anchor structure is processed. In most cells, an acyl chain modified to the inositol of GPI is removed by a GPI-inositol deacylase, PGAP1, in the ER. Inositol-deacylated GPI-APs become sensitive to bacterial phosphatidylinositol-specific phospholipase C (PI-PLC). We previously reported that GPI-APs are partially resistant to PI-PLC when PGAP1 activity is weakened by the deletion of selenoprotein T (SELT) or cleft lip and palate transmembrane protein 1 (CLPTM1). In this study, we found that the loss of TMEM41B, an ER-localized lipid scramblase, restored PI-PLC sensitivity of GPI-APs in SELT-knockout (KO) and CLPTM1-KO cells. In TMEM41B-KO cells, the transport of GPI-APs as well as transmembrane proteins from the ER to the Golgi was delayed. Furthermore, the turnover of PGAP1, which is mediated by ER-associated degradation, was slowed in TMEM41B-KO cells. Taken together, these findings indicate that inhibition of TMEM41B-dependent lipid scrambling promotes GPI-AP processing in the ER through PGAP1 stabilization and slowed protein trafficking.

研究领域: 细胞生物学 · 分子生物学 · 蛋白质运输 · 脂质代谢

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In vitro and in vivo assay of the ER lipid scramblase TMEM41B

A lipid scramblase TMEM41B is involved in the processing and transport of GPI-anchored proteins