Galperin Emilia, Sorkin Alexander
Department of Pharmacology, University of Colorado Health Sciences Center, Denver, CO 80262, USA.
J Cell Sci. 2003 Dec 1;116(Pt 23):4799-810. doi: 10.1242/jcs.00801.
Rab5 is a small GTPase that controls endocytosis and early endosome dynamics. To visualize active, GTP-loaded Rab5 in living cells, we developed molecular sensors consisting of the Rab5-binding fragments of Rabaptin5 or EEA.1 fused to yellow fluorescent protein (YFP). Interaction of these sensors with GTP-bound Rab5 fused to cyan fluorescent protein (CFP) resulted in fluorescence resonance energy transfer (FRET) between CFP and YFP. Activated Rab5 was detected by FRET microscopy in endosomal compartments and often concentrated in microdomains in the endosomal membrane. Although the plasma membrane-localized activity of Rab5 was not detected by light microscopy, overexpression of a GDP-bound mutant of CFP-Rab5(S34N) inhibited internalization of the epidermal growth factor receptor by retaining receptors in clathrin-coated pits. To test whether the Rab5(S34N) mutant affects endocytosis directly at the plasma membrane, CFP-Rab5 was fused to the plasma membrane targeting sequence of K-Ras containing a CAAX motif. The resulting chimeric CFP-Rab5-CAAX was located mainly in the plasma membrane and was capable of binding GTP as judged by FRET microscopy with the Rabaptin5-based sensor. Interestingly, EEA.1 sensor did not follow activated Rab5-CAAX to the plasma membrane, suggesting that the interaction of EEA.1 with Rab5 plays a secondary role in EEA.1 targeting. Overexpression of CFP-Rab5(S34N)CAAX prevented endocytosis of receptors by retaining them in coated pits. These data suggest that the dominant-negative effect of the Rab5(S34N) mutant on the late stages of endocytosis can be mediated through the inhibition of cytosol-associated or plasma-membrane-associated rather than endosome-associated regulators of Rab proteins.
Rab5是一种小GTP酶，可控制内吞作用和早期内体动力学。为了在活细胞中可视化活性的、负载GTP的Rab5，我们开发了由Rabaptin5或EEA.1的Rab5结合片段与黄色荧光蛋白（YFP）融合而成的分子传感器。这些传感器与融合了青色荧光蛋白（CFP）的GTP结合型Rab5相互作用，导致CFP和YFP之间发生荧光共振能量转移（FRET）。通过FRET显微镜在内体区室中检测到活化的Rab5，其通常集中在内体膜的微结构域中。尽管通过光学显微镜未检测到Rab5在质膜上的定位活性，但CFP-Rab5（S34N）的GDP结合突变体的过表达通过将表皮生长因子受体保留在网格蛋白包被小窝中而抑制了其内化。为了测试Rab5（S34N）突变体是否直接在质膜上影响内吞作用，将CFP-Rab5与含有CAAX基序的K-Ras的质膜靶向序列融合。通过基于Rabaptin5的传感器进行FRET显微镜观察判断，所得的嵌合CFP-Rab5-CAAX主要位于质膜中，并且能够结合GTP。有趣的是，EEA.1传感器并未跟随活化的Rab5-CAAX到达质膜，这表明EEA.1与Rab5的相互作用在EEA.1靶向中起次要作用。CFP-Rab5（S34N）CAAX的过表达通过将受体保留在包被小窝中而阻止了受体的内吞作用。这些数据表明，Rab5（S34N）突变体对内吞作用后期的显性负效应可通过抑制Rab蛋白的胞质溶胶相关或质膜相关而非内体相关调节因子来介导。
