Tisdale E J, Balch W E
Departments of Cell and Molecular Biology, The Scripps Research Institute, La Jolla, California 92037, USA.
J Biol Chem. 1996 Nov 15;271(46):29372-9. doi: 10.1074/jbc.271.46.29372.
The small GTPase Rab2 is a resident of pre-Golgi intermediates and required for protein transport from the endoplasmic reticulum (ER) to the Golgi complex (Tisdale, E. J., Bourne, J. R., Khosravi-Far, R. , Der, C. J., and Balch, W. E. (1992) J. Cell Biol. 119, 749-761). The Rab2 protein, like all small GTPases, contains conserved GTP-binding domains as well as hypervariable carboxyl-terminal and amino-terminal domains. While the role of the carboxyl terminus in specific membrane localization is well recognized, the potential role of the variable NH2 terminus remains to be clarified. To determine whether the NH2 terminus of Rab2 was required for its activity in vivo, a trans dominant mutant of Rab2 that inhibits ER to Golgi transport was progressively truncated and analyzed for its effect on vesicular stomatitis virus glycoprotein transport in a vaccinia-based transient expression system. Deletion of the first 14 amino-terminal residues resulted in the loss of the inhibitory properties of the mutant without affecting its post-translational processing or membrane association. To assess the potential role of the NH2 terminus in Rab2 function, a peptide corresponding to the first 13 amino acids following the initiator methionine was introduced into an in vitro assay that efficiently reconstitutes transport of vesicular stomatitis virus glycoprotein from the ER to the Golgi stack. This peptide was a potent inhibitor of transport. Biochemical and morphological studies revealed that the peptide strongly interfered with assembly of pre-Golgi intermediates which mediate segregation of anterograde and retrograde transported proteins en route to the Golgi. The combined results suggest that the NH2 terminus of Rab2 is required for its function and for direct interaction with components of the transport machinery involved in the maturation of pre-Golgi intermediates.
小GTP酶Rab2是高尔基体前体中间体的驻留蛋白，是内质网（ER）到高尔基体复合体蛋白质运输所必需的（蒂斯代尔，E.J.，伯恩，J.R.，霍斯拉维 - 法尔，R.，德尔，C.J.，和巴尔奇，W.E.（1992年）《细胞生物学杂志》119卷，749 - 761页）。Rab2蛋白与所有小GTP酶一样，包含保守的GTP结合结构域以及高度可变的羧基末端和氨基末端结构域。虽然羧基末端在特定膜定位中的作用已得到充分认识，但可变氨基末端的潜在作用仍有待阐明。为了确定Rab2的氨基末端在体内活性是否必需，对抑制ER到高尔基体运输的Rab2反式显性突变体进行逐步截短，并在基于痘苗病毒的瞬时表达系统中分析其对水疱性口炎病毒糖蛋白运输的影响。删除前14个氨基末端残基导致突变体的抑制特性丧失，而不影响其翻译后加工或膜结合。为了评估氨基末端在Rab2功能中的潜在作用，将对应于起始甲硫氨酸后前13个氨基酸的肽引入体外测定中，该测定有效地重建了水疱性口炎病毒糖蛋白从ER到高尔基体堆叠的运输。该肽是运输的有效抑制剂。生化和形态学研究表明，该肽强烈干扰高尔基体前体中间体的组装，高尔基体前体中间体介导在前往高尔基体途中顺行和逆行运输蛋白的分离。综合结果表明，Rab2的氨基末端是其功能所必需的，并且对于与参与高尔基体前体中间体成熟的运输机制成分的直接相互作用也是必需的。
