Chen Xuequn, Ernst Stephen A, Williams John A
Department of Molecular and Integrative Physiology, The University of Michigan, Ann Arbor, Michigan 48109, USA.
J Biol Chem. 2003 Dec 12;278(50):50053-60. doi: 10.1074/jbc.M309910200. Epub 2003 Sep 30.
Two dominant negative mutants of Rab3D, N135I and T36N were recently reported to inhibit the early phase of regulated amylase secretion from mouse pancreatic acini (Chen, X., Edwards, J. A., Logsdon, C. D., Ernst, S. A., and Williams, J. A. (2002) J. Biol. Chem. 277, 18002-18009). Immunocytochemical studies showed that while the wild-type Rab3D localized to zymogen granules, the two dominant negative mutants did not localize to granules and were primarily in the basolateral regions of the cell. The present study, therefore, evaluated the potential mechanisms by which the dominant negative mutants might act. An affinity precipitation assay based on the property of the Rab3 effector Rim1 to interact only with GTP-bound Rab3D was developed. 78.9 +/- 4.5% of total endogenous Rab3D was found in the GTP-bound form. Overexpression of HA-tagged Rab3D, and its Q81L, N135I, and T36N mutants had no effect on the total amount of endogenous Rab3D. However, the dominant negative mutants, T36N and N135I, reduced GTP-bound endogenous Rab3D by 70.0 +/- 3.5% and 72.7 +/- 1.2%, respectively, while the wild-type Rab3D and Q81L mutant had no effect. Triton X-114 phase separation and cell fractionation studies showed that dominant negative Rab3D mutants did not alter isoprenylation or membrane association of endogenous Rab3D. The dominant negative Rab3D did not affect the amount of endogenous Rab3D on purified zymogen granules as assessed by either Western blotting or immunocytochemistry, but reduced the GTP-bound form by 78.6 +/- 3.3%. The two dominant negative Rab3D mutants, therefore, interfere with endogenous Rab3D function by blocking the GDP/GTP exchange but not zymogen granule targeting of endogenous Rab3D.
最近有报道称，Rab3D的两种显性负性突变体N135I和T36N可抑制小鼠胰腺腺泡中受调节的淀粉酶分泌的早期阶段（Chen, X., Edwards, J. A., Logsdon, C. D., Ernst, S. A., and Williams, J. A. (2002) J. Biol. Chem. 277, 18002 - 18009）。免疫细胞化学研究表明，野生型Rab3D定位于酶原颗粒，而这两种显性负性突变体不定位于颗粒，主要位于细胞的基底外侧区域。因此，本研究评估了显性负性突变体可能发挥作用的潜在机制。基于Rab3效应器Rim1仅与GTP结合的Rab3D相互作用的特性，开发了一种亲和沉淀测定法。发现78.9±4.5%的内源性Rab3D以GTP结合形式存在。HA标签的Rab3D及其Q81L、N135I和T36N突变体的过表达对内源性Rab3D的总量没有影响。然而，显性负性突变体T36N和N135I分别使GTP结合的内源性Rab3D减少了70.0±3.5%和72.7±1.2%，而野生型Rab3D和Q81L突变体则没有影响。Triton X - 114相分离和细胞分级分离研究表明，显性负性Rab3D突变体不会改变内源性Rab3D的异戊二烯化或膜结合。通过蛋白质印迹法或免疫细胞化学评估，显性负性Rab3D对纯化的酶原颗粒上内源性Rab3D的量没有影响，但使GTP结合形式减少了78.6±3.3%。因此，这两种显性负性Rab3D突变体通过阻断GDP / GTP交换而非内源性Rab3D的酶原颗粒靶向作用来干扰内源性Rab3D的功能。
