Wanschers Bas, van de Vorstenbosch Rinske, Wijers Mietske, Wieringa Bé, King Stephen M, Fransen Jack
Department of Cell Biology, Nijmegen Centre for Molecular Life Sciences, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands.
Cell Motil Cytoskeleton. 2008 Mar;65(3):183-96. doi: 10.1002/cm.20254.
The small GTPase Rab6 is a key regulator in the retrograde transfer from endosomes via the Golgi to the ER. Three isoforms of Rab6 have been identified, the ubiquitously expressed Rab6A and Rab6A', and the brain specific Rab6B. Recent studies have shown that Rab6A' is the major isoform regulating this retrograde transport. Cytoplasmic dynein is the main motor protein complex for this transport. Dynein consists of two heavy chains, two intermediate chains, four light intermediate chains and several light chains, called roadblock/LC7 proteins or DYNLRB proteins. In mammalian cells two light chain isoforms have been identified, DYNLRB1 and DYNLRB2. We here show with yeast-two-hybrid, co-immunoprecipitation and pull down studies that DYNLRB1 specifically interacts with all three Rab6 isoforms and co-localises at the Golgi. This is the first example of a direct interaction between Rab6 isoforms and the dynein complex. Pull down experiments showed further preferred association of DYNLRB1 with GTP-bound Rab6A and interestingly GDP-bound Rab6A' and Rab6B. In addition DYNLRB1 was found in the Golgi apparatus where it co-localises with EYFP-Rab6 isoforms. DYNLRB is a putative modulator of the intrinsic GTPase activity of GTP-binding proteins. In vitro we were not able to reproduce this effect on Rab6 GTPase activity.
小GTP酶Rab6是从内体经高尔基体到内质网逆行转运的关键调节因子。已鉴定出Rab6的三种同工型，即广泛表达的Rab6A和Rab6A'，以及脑特异性的Rab6B。最近的研究表明，Rab6A'是调节这种逆行转运的主要同工型。胞质动力蛋白是这种转运的主要动力蛋白复合体。动力蛋白由两条重链、两条中间链、四条轻中间链和几条轻链组成，这些轻链被称为路障/LC7蛋白或DYNLRB蛋白。在哺乳动物细胞中已鉴定出两种轻链同工型，即DYNLRB1和DYNLRB2。我们在此通过酵母双杂交、免疫共沉淀和下拉实验表明，DYNLRB1与所有三种Rab6同工型特异性相互作用，并在高尔基体中共定位。这是Rab6同工型与动力蛋白复合体之间直接相互作用的首个例子。下拉实验进一步表明，DYNLRB1与GTP结合的Rab6A以及有趣的是与GDP结合的Rab6A'和Rab6B有更优先的结合。此外，在高尔基体中发现了DYNLRB1，它与EYFP-Rab6同工型共定位。DYNLRB是一种假定的GTP结合蛋白内在GTP酶活性的调节剂。在体外，我们无法重现这种对Rab6 GTP酶活性的影响。
