16.4
核定位信号和输入
靶向细胞核中的蛋白质会携带一段较短的氨基酸序列，并将其称为核定位信号（NLS）。经典的核定位信号有两种类型：单分型经典核定位信号和双分型经典核定位信号。单分型经典核定位信号（cNLS）是由 4-8 个氨基酸中的单个簇所组成的。双分型经典核定位信号则是由两个 2-3 个氨基酸中的簇和桥接两个簇的 9-…
从细胞质输入到细胞核的蛋白质含有短段氨基酸序列，称为核定位信号或NLS。
核输入蛋白（importins）是一类特化的核输入受体，可识别 cargo 蛋白的核定位信号（NLS），并通过核孔复合体（NPCs）将其转运至细胞核内。
输入蛋白以可溶性异源二聚体形式存在于细胞质中，由α和β亚基组成。输入蛋白α亚基结合 cargo 蛋白的核定位信号（NLS），形成 cargo-受体复合物。
Importin β 结合从核孔复合体（NPC）延伸出的细胞质纤丝，并将货物-受体复合物停靠在通道的开口处。
核孔复合体的内通道内衬有富含苯丙氨酸-甘氨酸（phenylalanine-glycine）或FG重复序列，可形成类似凝胶的选择性屏障。
当货物-受体复合物通过通道时，β亚基会与FG重复序列形成多个弱相互作用，并在通道上跳跃，打破FG重复序列之间的相互作用，从而溶解凝胶样屏障。通过这种反复的接触与溶解过程，货物-受体复合物得以进入细胞核内部。
在细胞核内，一种称为Ran的GTP结合蛋白与importin beta结合，并诱导该受体发生构象变化，从而释放 cargo 蛋白。
随后，importin-Ran-GTP复合物被转运回细胞质，在那里GTP被水解，释放出importin，以便进行下一轮的货物输入。
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Q1: What are nuclear localization signals and why do proteins need them?
Nuclear localization signals (NLS) are short stretches of amino acid sequences that mark proteins for nuclear import. Proteins destined for the nucleus contain these signals, which are recognized by specialized import receptors called importins. Without NLS, proteins cannot be directed to and transported across the nuclear envelope.
Q2: How do importin alpha and importin beta work together during nuclear import?
Importin alpha recognizes and binds the NLS of cargo proteins, forming a cargo-receptor complex. Importin beta then binds to cytoplasmic fibrils extending from the nuclear pore complex and docks the cargo-receptor complex at the channel opening. Together, these subunits facilitate transport of the cargo protein into the nucleus.
Q3: What role do FG-rich repeats play in nuclear pore transport?
The inner channel of the nuclear pore complex is lined with phenylalanine-glycine (FG) repeats that form a gel-like selective barrier. As the cargo-receptor complex moves through, the importin beta subunit makes weak contacts with these FG repeats and hops across the channel, dissolving the gel-like barrier and allowing passage of the cargo protein.
Q4: What are the different types of classical nuclear localization signals?
Classical NLS exist in two forms: monopartite and bipartite. Monopartite NLS consists of a single cluster of 4-8 amino acids rich in lysine and arginine. Bipartite NLS contains two clusters of 2-3 amino acids separated by a 9-12 residue proline-rich linker. Both types are recognized by importin alpha for nuclear targeting.
Q5: How does Ran-GTP release cargo proteins inside the nucleus?
Once inside the nucleus, the GTP-bound protein Ran binds to importin beta and induces a conformational change in the receptor. This conformational change causes the importin alpha-cargo complex to dissociate, releasing the cargo protein into the nucleus. The importin-Ran-GTP complex is then transported back to the cytosol for recycling.
Q6: What happens to importins after they return to the cytosol?
When the importin-Ran-GTP complex returns to the cytosol, GTP is hydrolyzed to GDP, releasing the importins. The freed importin alpha and beta subunits dissociate and become available as soluble dimers in the cytosol for another round of cargo import, enabling continuous nuclear protein transport.
Q7: What are non-classical and cryptic nuclear localization signals?
Beyond classical monopartite and bipartite NLS, cells use non-classical NLS, putative NLS, spatial epitope NLS, and cryptic NLS for nuclear targeting. These alternative signal types provide flexibility in protein recognition and allow importins to transport diverse cargo proteins. Some proteins contain multiple NLS to enhance nuclear import efficiency.