Navarro-Lérida Inmaculada, Martínez Moreno Mónica, Roncal Fernando, Gavilanes Francisco, Albar Juan Pablo, Rodríguez-Crespo Ignacio
Departamento de Bioquímicay Biología Molecular, Facultad de Ciencias Químicas, Universidad Complutense de Madrid, Madrid, Spain.
Proteomics. 2004 Feb;4(2):339-46. doi: 10.1002/pmic.200300528.
Cytoplasmic dynein is a large minus end-directed microtubule motor that translocates cargos towards the minus end of microtubules. Light chain 8 of the dynein machinery (LC8) has been reported to interact with a large variety of proteins that possess K/RSTQT or GIQVD motifs in their sequence, hence permitting their transport in a retrograde manner. Yeast two-hybrid analysis has revealed that in brain, LC8 associates directly with several proteins such as neuronal nitric oxide synthase, guanylate kinase domain-associated protein and gephyrin. In this work, we report the identification of over 40 polypeptides, by means of a proteomic approach, that interact with LC8 either directly or indirectly. Many of the neuronal proteins that we identified cluster at the post-synaptic terminal, and some of them such as phosphofructokinase, lactate dehydrogenase or aldolase are directly involved in glutamate metabolism. Other pool of proteins identified displayed the LC8 consensus binding motif. Finally, recombinant LC8 was produced and a library of overlapping dodecapeptides (pepscan) was employed to map the LC8 binding site of some of the proteins that were previously identified using the proteomic approach, hence confirming binding to the consensus binding sites.
细胞质动力蛋白是一种大型的向微管负端移动的微管马达蛋白，可将货物向微管的负端转运。动力蛋白机制的轻链8（LC8）已被报道可与多种在其序列中具有K/RSTQT或GIQVD基序的蛋白质相互作用，从而允许它们以逆行方式运输。酵母双杂交分析表明，在大脑中，LC8直接与几种蛋白质相关联，如神经元型一氧化氮合酶、鸟苷酸激酶结构域相关蛋白和gephyrin。在这项工作中，我们报告了通过蛋白质组学方法鉴定出40多种与LC8直接或间接相互作用的多肽。我们鉴定出的许多神经元蛋白聚集在突触后末端，其中一些蛋白，如磷酸果糖激酶、乳酸脱氢酶或醛缩酶，直接参与谷氨酸代谢。鉴定出的其他蛋白质组显示出LC8共有结合基序。最后，制备了重组LC8，并使用重叠十二肽文库（肽扫描）来绘制一些先前使用蛋白质组学方法鉴定的蛋白质的LC8结合位点，从而证实与共有结合位点的结合。
