Nuclear pore targeting of the yeast Pom33 nucleoporin depends on karyopherin and lipid binding
Nuclear pore targeting of the yeast Pom33 nucleoporin depends on karyopherin and lipid binding
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DOI：
10.1242/jcs.158915
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发表时间：
2015-01-15
影响因子：
4
通讯作者：
Doye, Valerie
中科院分区：
文献类型：
--
作者：
Floch, Aurelie G.;Tareste, David;Doye, Valerie
Pom 33是酵母核孔复合体（NPC）的一种膜蛋白，是NPC正常分布和组装所必需的。为了表征Pom 33 NPC靶向决定簇，我们进行了免疫沉淀实验，然后进行质谱分析。这确定了一个新的Pom 33伙伴，核输入因子Kap 123。体外实验揭示了Pom 33 C-末端结构域（CTD）和Kap 123之间的直接相互作用。计算机模拟分析预测Pom 33-CTD内存在两个两亲性α-螺旋。圆二色性和脂质体共浮选分析表明，该结构域能够折叠成α-螺旋在脂质体的存在下，并优先结合到高度弯曲的脂质膜。当在酵母中表达时，在消除Pom 33-CTD膜结合的条件下，该结构域表现为Kap 123依赖性核定位信号（NLS）。尽管Pom 33 C-末端结构域（Pom（Delta DCTD）-GFP）的缺失损害了Pom 33的稳定性和NPC靶向，但影响Kap 123结合或α-螺旋的两亲性质的突变体没有显示出任何可检测的缺陷。然而，脂质和Kap 123结合的组合损伤影响Pom 33靶向NPC。这些数据突出了Pom 33的适当NPC定位的多个决定因素和机制的要求。
Pom33 is an integral membrane protein of the yeast nuclear pore complex (NPC), and it is required for proper NPC distribution and assembly. To characterize the Pom33 NPC-targeting determinants, we performed immunoprecipitation experiments followed by mass spectrometry analyses. This identified a new Pom33 partner, the nuclear import factor Kap123. In vitro experiments revealed a direct interaction between the Pom33 C-terminal domain (CTD) and Kap123. In silico analysis predicted the presence of two amphipathic alpha-helices within Pom33-CTD. Circular dichroism and liposome co-flotation assays showed that this domain is able to fold into alpha-helices in the presence of liposomes and preferentially binds to highly curved lipid membranes. When expressed in yeast, under conditions abolishing Pom33-CTD membrane association, this domain behaves as a Kap123-dependent nuclear localization signal (NLS). Although deletion of Pom33 C-terminal domain (Pom(Delta DCTD)-GFP) impaired Pom33 stability and NPC targeting, mutants affecting either Kap123 binding or the amphipathic properties of the alpha-helices did not display any detectable defect. However, combined impairment of lipid and Kap123 binding affects targeting of Pom33 to NPCs. These data highlight the requirement of multiple determinants and mechanisms for proper NPC localization of Pom33.