Genome-wide CRISPR screen reveals CLPTM1L as a lipid scramblase required for efficient glycosylphosphatidylinositol biosynthesis.
Genome-wide CRISPR screen reveals CLPTM1L as a lipid scramblase required for efficient glycosylphosphatidylinositol biosynthesis.
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DOI：
10.1073/pnas.2115083119
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发表时间：
2022-04-05
影响因子：
11.1
通讯作者：
Kinoshite, Taroh
中科院分区：
文献类型：
--
作者：
Wang, Yicheng;Menon, Anant K.;Maki, Yuta;Yi-Shi Liu;Iwasaki, Yugo;Fujita, Morihisa;Guerrero, Paula A.;Silva, Daniel Varo'n;Seeberger, Peter H.;Murakami, Yoshiko;Kinoshite, Taroh
超燃酶在不消耗ATP的情况下通过脂质双分子层转运脂质，从而调节细胞膜中的脂质分布。细胞质溶胶到管腔的跨内质网（ER）膜易位是真核生物中参与翻译后蛋白修饰的脂糖缀合物的共同过程。这些易位被认为是由特定的内质网转录酶介导的，但这些蛋白质的身份和潜在的分子机制一直是难以捉摸的。本研究表明，具有8个跨膜结构域的完整膜蛋白CLPTM1L是内质网膜中参与高效糖基磷脂酰肌醇生物合成的主要脂质合成酶。我们的研究结果验证了长期存在的假设，即脂质超燃酶确保脂质糖缀合物在蛋白质糖基化途径中通过内质网膜有效易位。糖基磷脂酰肌醇（gpi）是一种复杂的糖脂，作为许多真核细胞表面蛋白的膜锚定物。GPIs的生物合成是在内质网（ER）的细胞质面通过生成n -乙酰氨基葡萄糖-磷脂酰肌醇（GlcNAc-PI）开始的。第二种中间体，葡萄糖氨基-磷脂酰肌醇（GlcN-PI），通过膜转移到管腔表面，用于随后的生物合成步骤和附着在蛋白质上。gln - pi的腔内易位机制尚不清楚。在此，我们报道了在将化学合成的GlcNAc-PI添加到不能合成GlcNAc-PI的piga敲除细胞后，挽救gpi锚定蛋白表达所需的全基因组CRISPR敲除筛选。我们鉴定了一种内质网多通膜蛋白CLPTM1L（唇腭裂跨膜蛋白1样）是GPIs高效生物合成所需的GlcN-PI重组酶。在piga敲除细胞中敲除CLPTM1L会影响化学合成的GlcNAc-PI和GlcN-PI对GPI生物合成的有效利用。纯化的CLPTM1L在体外对GlcN-PI、GlcNAc-PI、PI和其他几种磷脂进行了搅和。CLPTM1L是PQ-loop蛋白家族的一员，代表了一种与已知脂质重组酶没有结构相似性的脂质重组酶。敲除各种野生型哺乳动物培养细胞中的CLPTM1L可部分降低gpi锚定蛋白的水平。这些结果表明，CLPTM1L是参与GlcN-PI通过内质网膜向细胞质到腔内转运的主要脂质合成酶，以实现高效的GPI生物合成。
Scramblases translocate lipids across the lipid bilayer without consumption of ATP, thereby regulating lipid distributions in cellular membranes. Cytosol-to-lumen translocation across the endoplasmic reticulum (ER) membrane is a common process among lipid glycoconjugates involved in posttranslational protein modifications in eukaryotes. These translocations are thought to be mediated by specific ER-resident scramblases, but the identity of these proteins and the underlying molecular mechanisms have been elusive. Here, we show that CLPTM1L, an integral membrane protein with eight putative transmembrane domains, is the major lipid scramblase involved in efficient glycosylphosphatidylinositol biosynthesis in the ER membrane. Our results validate the long-standing hypothesis that lipid scramblases ensure the efficient translocations of lipid glycoconjugates across the ER membrane for protein glycosylation pathways. Glycosylphosphatidylinositols (GPIs) are complex glycolipids that act as membrane anchors of many eukaryotic cell surface proteins. Biosynthesis of GPIs is initiated at the cytosolic face of the endoplasmic reticulum (ER) by generation of N-acetylglucosaminyl-phosphatidylinositol (GlcNAc-PI). The second intermediate, glucosaminyl-phosphatidylinositol (GlcN-PI), is translocated across the membrane to the luminal face for later biosynthetic steps and attachment to proteins. The mechanism of the luminal translocation of GlcN-PI is unclear. Here, we report a genome-wide CRISPR knockout screen of genes required for rescuing GPI-anchored protein expression after addition of chemically synthesized GlcNAc-PI to PIGA-knockout cells that cannot synthesize GlcNAc-PI. We identified CLPTM1L (cleft lip and palate transmembrane protein 1-like), an ER-resident multipass membrane protein, as a GlcN-PI scramblase required for efficient biosynthesis of GPIs. Knockout of CLPTM1L in PIGA-knockout cells impaired the efficient utilization of chemically synthesized GlcNAc-PI and GlcN-PI for GPI biosynthesis. Purified CLPTM1L scrambled GlcN-PI, GlcNAc-PI, PI, and several other phospholipids in vitro. CLPTM1L, a member of the PQ-loop family of proteins, represents a type of lipid scramblase having no structural similarity to known lipid scramblases. Knockout of CLPTM1L in various wild-type mammalian cultured cells partially decreased the level of GPI-anchored proteins. These results suggest that CLPTM1L is the major lipid scramblase involved in cytosol-to-lumen translocation of GlcN-PI across the ER membrane for efficient GPI biosynthesis.
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DOI：
10.1083/jcb.202103105
发表时间：
2021-06-07
期刊：
The Journal of cell biology
影响因子：
--
作者：
Li YE;Wang Y;Du X;Zhang T;Mak HY;Hancock SE;McEwen H;Pandzic E;Whan RM;Aw YC;Lukmantara IE;Yuan Y;Dong X;Don A;Turner N;Qi S;Yang H
通讯作者：
Yang H
影响因子：
4
作者：
Guerrero PA;Murakami Y;Malik A;Seeberger PH;Kinoshita T;Varón Silva D
通讯作者：
Varón Silva D
影响因子：
7.8
作者：
Vidugiriene, J;Menon, A K
通讯作者：
Menon, A K
影响因子：
2.9
作者：
Kol, MA;de Kroon, AIPM;de Kruijff, B
通讯作者：
de Kruijff, B
影响因子：
20.3
作者：
Kawagoe, K;Kitamura, D;Takeda, J
通讯作者：
Takeda, J