Beuttenmüller M, Chen M, Janetzko A, Kühn S, Traub P
Max-Planck-Institut für Zelbiologie, Ladenburg/Heidelberg, Germany.
Exp Cell Res. 1994 Jul;213(1):128-42. doi: 10.1006/excr.1994.1182.
The biological functions of the non-alpha-helical, N- and C-terminal head and tail domains of intermediate filament (IF) proteins are still ill-defined. Previously, it has been shown that the basic, N-terminal head piece of the type III IF protein vimentin is essential for regular IF assembly and that arginine residues within the N-terminus may be involved. In order to identify particular regions within this domain essential for filament formation and stabilization, N-terminally truncated and arginine substitution forms of vimentin were constructed via site-directed in vitro mutagenesis of murine vimentin cDNA. The de novo filament assembly properties of these modified forms were compared with those of wild-type vimentin after transient expression in vimentin-free, cultured cells. In order to investigate their filament assembly competence in vitro, they were also produced in an E. coli expression system. It could be demonstrated that deletion of the first 10, 13, 17, and 32 amino acid residues, respectively, from the N-terminus of vimentin has an increasingly deleterious effect on filament assembly in vitro and network formation in vivo and that, thus, the highly conserved sequence motif, SSYRRXFGG, located in the N-terminus of various IF proteins and partially or totally removed by the above deletions plays a particularly important role in both activities. These results were confirmed and extended by arginine point mutations in the N-terminal head region, which showed that only one of the two adjacent arginine residues located within the conserved sequence motif is essential for filament assembly and stability in vitro as well as network formation in vivo. The neighboring arginine residues could be replaced by lysine residues without severe effects on the assembly properties of the respective mutant proteins. Distinction between the assembly-promoting potentials of the two arginine residues of the N-terminal doublet was considerably facilitated by a Val389-->Asp substitution toward the carboxy-end of the 2B segment of the vimentin rod domain. The synergistic effect of point mutations in this and the N-terminal region of the vimentin molecule implies the interaction of both protein domains in the process of filament assembly. Mutant vimentin proteins that were characterized by distinct incompetence to assemble into IFs caused a massive collapse of the endogenous vimentin filament system when expressed in mouse skin fibroblasts.
中间丝（IF）蛋白的非α螺旋、N端和C端头部及尾部结构域的生物学功能仍不明确。此前研究表明，III型IF蛋白波形蛋白的碱性N端头部片段对于正常的IF组装至关重要，且N端的精氨酸残基可能参与其中。为了确定该结构域内对丝形成和稳定至关重要的特定区域，通过对小鼠波形蛋白cDNA进行定点体外诱变构建了N端截短和精氨酸替代形式的波形蛋白。在无波形蛋白的培养细胞中瞬时表达后，将这些修饰形式的从头丝组装特性与野生型波形蛋白的进行比较。为了研究它们在体外的丝组装能力，还在大肠杆菌表达系统中进行了表达。结果表明，分别从波形蛋白N端缺失前10、13、17和32个氨基酸残基，对体外丝组装和体内网络形成的有害影响越来越大，因此，位于各种IF蛋白N端、部分或全部被上述缺失去除的高度保守序列基序SSYRRXFGG在这两种活性中起特别重要的作用。N端头部区域的精氨酸点突变证实并扩展了这些结果，表明位于保守序列基序内的两个相邻精氨酸残基中只有一个对体外丝组装和稳定性以及体内网络形成至关重要。相邻的精氨酸残基可以被赖氨酸残基取代，而对相应突变蛋白的组装特性没有严重影响。波形蛋白杆状结构域2B段羧基端的Val389→Asp取代极大地促进了对N端双峰中两个精氨酸残基组装促进潜力的区分。该区域和波形蛋白分子N端的点突变的协同效应意味着这两个蛋白结构域在丝组装过程中相互作用。以明显无法组装成IF为特征的突变波形蛋白在小鼠皮肤成纤维细胞中表达时，会导致内源性波形蛋白丝系统大量崩溃。
