Molecular consequences of SCA5 mutations in the spectrin-repeat domains of β-III-spectrin

Spinocerebellar ataxia type 5 (SCA5) mutations in the protein β-III-spectrin cluster to the N-terminal actin-binding domain (ABD) and the central spectrin-repeat domains (SRDs). We previously reported that a common molecular consequence of ABD-localized SCA5 mutations is increased actin binding. However, little is known about the molecular consequences of the SRD-localized mutations. It is known that the SRDs of β-spectrin proteins interact with α-spectrin to form an α/β-spectrin dimer. In addition, it is known that SRDs neighboring the β-spectrin ABD enhance actin binding. Here, we tested the impact of the SRD-localized R480W and E532_M544del mutations on the binding of β-III-spectrin to α-II-spectrin and actin. R480W is associated with a severe infantile onset form of SCA5, while E532_M544del is associated with milder symptoms that begin in adulthood. We show that both the R480W and E532_M544del mutants can bind α-II-spectrin. However, E532_M544del causes partial uncoupling of complementary SRDs in the α/β-spectrin dimer. Further, the R480W mutant forms large intracellular inclusions when coexpressed with α-II-spectrin in cells, supporting that R480W grossly disrupts the α-II/β-III-spectrin complex. Moreover, actin-binding assays show that E532_M544del, but not R480W, increases β-III-spectrin actin binding. Additionally, we demonstrate that R480W α-II/β-III-spectrin inclusions contain F-actin, accumulate the spectrin-binding protein ankyrin-R, and localize immediately adjacent to the Golgi complex. Two additional infantile onset mutations, R437W and R437Q, but not the adult onset T472M mutation, also cause formation of large α-II/β-III-spectrin inclusions. We suggest that the intracellular inclusions caused by R480W, R437W, and R437Q drive the more severe disease symptoms associated with these mutations.