Basic amino acid residue cluster within nuclear targeting sequence motif is essential for cytoplasmic plectin-vimentin network junctions.
Basic amino acid residue cluster within nuclear targeting sequence motif is essential for cytoplasmic plectin-vimentin network junctions.
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DOI：
10.1083/jcb.134.6.1455
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发表时间：
1996-09
期刊：
影响因子：
--
通讯作者：
Wiche G
中科院分区：
文献类型：
--
作者：
Nikolic B;Mac Nulty E;Mir B;Wiche G
我们已经产生了一系列的plectin缺失和突变的cdna结构，以剖析介导plectin与中间丝(IF)网络相互作用的功能序列，并对它们在大鼠袋鼠PtK2细胞中异位表达时协同排列或破坏中间丝的能力进行了评分。我们发现，在plectin的羧基末端重复序列5结构域中，大约50个氨基酸残基是PtK2细胞的波形蛋白和细胞角蛋白IF网络的唯一结合位点。IF结合域的一部分被发现构成了一个功能性核定位信号(NLS)基序，与该序列相关的细胞质蛋白的核输入证明了这一点。定点突变揭示了位于NLS序列基序中的四个碱性氨基酸残基(arg4277-arg4280)的特定簇对于IF结合是必不可少的。当相应于PtK2细胞表达的突变蛋白在细菌中表达时，纯化的蛋白进行了Eu3+标记的波形蛋白灵敏的定量重叠结合分析，测得的突变蛋白的相对结合量与在活细胞中观察到的突变蛋白的表型完全一致。利用重组蛋白，我们还通过负染色和旋转阴影电子显微镜显示，在体外组装的波形蛋白中间丝在与plectin Repeat 5结构域孵育时形成致密的聚集体，而不是Repeat 4结构域或突变的Repeat 5结构域。
We have generated a series of plectin deletion and mutagenized cDNA constructs to dissect the functional sequences that mediate plectin's interaction with intermediate filament (IF) networks, and scored their ability to coalign or disrupt intermediate filaments when ectopically expressed in rat kangaroo PtK2 cells. We show that a stretch of approximately 50 amino acid residues within plectin's carboxy-terminal repeat 5 domain serves as a unique binding site for both vimentin and cytokeratin IF networks of PtK2 cells. Part of the IF-binding domain was found to constitute a functional nuclear localization signal (NLS) motif, as demonstrated by nuclear import of cytoplasmic proteins linked to this sequence. Site directed mutagenesis revealed a specific cluster of four basic amino acid residues (arg4277-arg4280) residing within the NLS sequence motif to be essential for IF binding. When mutant proteins corresponding to those expressed in PtK2 cells were expressed in bacteria and purified proteins subjected to a sensitive quantitative overlay binding assay using Eu3+-labeled vimentin, the relative binding capacities of mutant proteins measured were fully consistent with the mutant's phenotypes observed in living cells. Using recombinant proteins we also show by negative staining and rotary shadowing electron microscopy that in vitro assembled vimentin intermediate filaments become packed into dense aggregates upon incubation with plectin repeat 5 domain, in contrast to repeat 4 domain or a mutated repeat 5 domain.
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影响因子：
4.2
作者：
ERRANTE, LD;WICHE, G;SHAW, G
通讯作者：
SHAW, G
影响因子：
19
作者：
ADAM, SA
通讯作者：
ADAM, SA
DOI：
10.1016/s0141-8130(05)80004-2
发表时间：
1992-06-01
影响因子：
8.2
作者：
GREEN, KJ;VIRATA, MLA;PARRY, DAD
通讯作者：
PARRY, DAD
影响因子：
3.7
作者：
GRAHAM, FL;VANDEREB, AJ
通讯作者：
VANDEREB, AJ
影响因子：
3.3
作者：
Foisner, R;Malecz, N;Wiche, G
通讯作者：
Wiche, G