Bananis Eustratios, Murray John W, Stockert Richard J, Satir Peter, Wolkoff Allan W
Marion Bessin Liver Research Center, Albert Einstein College of Medicine, Bronx, NY 10461, USA.
J Cell Sci. 2003 Jul 1;116(Pt 13):2749-61. doi: 10.1242/jcs.00478. Epub 2003 May 20.
We previously established conditions to reconstitute kinesin-dependent early endocytic vesicle motility and fission on microtubules in vitro. The present study examined the question whether motility and fission are regulated in this system. Screening for proteins by immunofluorescence microscopy revealed that the small G protein, Rab4, was associated with 80% of hepatocyte-derived early endocytic vesicles that contain the ligand asialoorosomucoid (ASOR). By contrast, other markers for early endocytic vesicles including clathrin, Rab5 and EEA1 were present in the preparation but did not colocalize with the ASOR vesicles. Guanine nucleotides exchanged into the Rab4 present on the vesicles as shown by solubilization of Rab4 by Rab-GDI; solubilization was inhibited by incubation with GTP-gamma-S and promoted by GDP. Pre-incubation of vesicles with GDP increased the number of vesicles moving on microtubules and markedly increased vesicle fission. This increase in motility from GDP was shown to be towards the minus end of microtubules, possibly through activation of the minus-end-directed kinesin, KIFC2. Pre-incubation of vesicles with GTP-gamma-S, by contrast, repressed motility. Addition of exogenous GST-Rab4- GTP-gamma-S led to a further repression of motility and fission. Repression was not seen with addition of GST-Rab4-GDP. Treatment of vesicles with Rab4 antibody also repressed motility, and repression was not seen when vesicles were pre-incubated with GDP. Based on these results we hypothesize that endogenous Rab4-GTP suppresses motility of ASOR-containing vesicles in hepatocytes and that conversion of Rab4-GTP to Rab4-GDP serves as a molecular switch that activates minus-end kinesin-based motility, facilitating early endosome fission and consequent receptor-ligand segregation.
我们之前建立了在体外重建驱动蛋白依赖的早期内吞囊泡在微管上运动和裂变的条件。本研究探讨了该系统中运动和裂变是否受到调控这一问题。通过免疫荧光显微镜筛选蛋白质发现，小G蛋白Rab4与80%含有配体去唾液酸糖蛋白（ASOR）的肝细胞来源的早期内吞囊泡相关。相比之下，早期内吞囊泡的其他标志物，包括网格蛋白、Rab5和EEA1在制剂中存在，但不与ASOR囊泡共定位。如通过Rab - GDI使Rab4溶解所显示的，鸟嘌呤核苷酸交换到囊泡上存在的Rab4中；用GTP -γ- S孵育可抑制溶解，而GDP则促进溶解。囊泡与GDP预孵育增加了在微管上移动的囊泡数量，并显著增加了囊泡裂变。GDP引起的这种运动增加显示是朝向微管的负端，可能是通过激活负端驱动的驱动蛋白KIFC2。相比之下，囊泡与GTP -γ- S预孵育会抑制运动。添加外源性GST - Rab4 - GTP -γ- S导致运动和裂变进一步受到抑制。添加GST - Rab4 - GDP则未观察到抑制作用。用Rab4抗体处理囊泡也会抑制运动，而当囊泡与GDP预孵育时则未观察到抑制作用。基于这些结果，我们推测内源性Rab4 - GTP抑制肝细胞中含ASOR囊泡的运动，并且Rab4 - GTP向Rab4 - GDP的转化作为一种分子开关，激活基于负端驱动蛋白的运动，促进早期内体裂变以及随后的受体 - 配体分离。
