Highly Purified Inner-Arm Dynein E from Chlamydomonas Reinhardtii Studied with in vitro Motility Assay

Green alga Chlamydomonas reinhardtii has two flagella at the front of the cell and beats them to swim. A flagellium is a biomolecular machine that consists of various proteins in a highly regulated manner. It is considered that an alignment of the various species of dynein proteins, which generate power using energy released upon ATP hydrolysis, plays important roles in the flagellum beating.Recently, great progress has been made in understanding of construction of a Chlamydomonas flagellum by the use of cryo-electron microscopic tomography. Dyneins are arranged in 96-nm periodic manner for a direction of a longitudinal axis of a flagellum. 8 species of dyneins are called as “inner-arm dyneins”. In 96-nm period, they make specific 4 pairs and are arranged in equally-spaced 4 places.Making use of in vitro motility assay, we can evaluate activities of dyneins as “microtubule sliding”. Dynein “c” slides microtubules relatively fast among the 8 inner-arms, and is well-studied. It has shown that dynein c pairs with dynein “e” in a flagellium. However, properties of dynein e are not known much. One reason for the difficulty is that anion exchange chromatography, a major purification method for inner arm dyneins, cannot separate dynein e and dynein d well.We obtained highly purified dynein e and c using two steps of anion exchange chromatography. We will present properties of dynein e and of a mixture of dynein e and c.