ZFYVE27 (SPG33), a novel spastin-binding protein, is mutated in hereditary spastic paraplegia.

Spastin, the most commonly mutated protein in the autosomal dominant form of hereditary spastic paraplegia (AD-HSP) has been suggested to be involved in vesicular cargo trafficking; however, a comprehensive function of spastin has not yet been elucidated. To characterize the molecular function of spastin, we used the yeast two-hybrid approach to identify new interacting partners of spastin. Here, we report ZFYVE27, a novel member of the FYVE-finger family of proteins, as a specific spastin-binding protein, and we validate the interaction by both in vivo coimmunoprecipitation and colocalization experiments in mammalian cells. More importantly, we report a German family with AD-HSP in which ZFYVE27 (SPG33) is mutated; furthermore, we demonstrate that the mutated ZFYVE27 protein shows an aberrant intracellular pattern in its tubular structure and that its interaction with spastin is severely affected. We postulate that this specific mutation in ZFYVE27 affects neuronal intracellular trafficking in the corticospinal tract, which is consistent with the pathology of HSP.

Spastin, the most commonly mutated protein in hereditary spastic paraplegia interacts with Reticulon 1 an endoplasmic reticulum protein.

Spastin, an ATPase belonging to the AAA family of proteins is most commonly mutated in autosomal dominant hereditary spastic paraplegias (HSP). Spastin is a multifaceted protein with versatile role in cellular events, principally involved in microtubule dynamics. To gain further insight into the molecular function of spastin, we used the yeast two-hybrid approach to identify novel interacting partners of spastin. Using spastin as bait, we identified reticulon 1 (RTN1) and reticulon 3 (RTN3) as potential spastin interacting proteins. RTN1 and RTN3 belong to the reticulon (RTN) gene family, which are primarily expressed in the endoplasmic reticulum. Moreover, RTN1 is known to play a role in vesicular transport processes. Using in vitro and in vivo immunoprecipitation experiments, we were able to demonstrate that RTN1 interacts specifically with spastin. Intracellular distribution studies using immunostaining and overexpression of epitope-tagged protein revealed an obvious colocalization of spastin and RTN1 in discrete vesicles in the cytoplasm. Spastin mediates its interaction with RTN1 through its N-terminal region containing a microtubule-interacting and trafficking domain. It is interesting to note that the aberrant intracellular distribution of a truncated spastin protein was rescued by coexpression with RTN1, which highlights the physiological significance of this interaction. Our findings strengthen the hypothesis that disruption of intracellular vesicular transport processes could cause HSP. It is interesting to note that RTN1 is localized to 14q23.1 where SPG15 locus was mapped. Therefore, we considered RTN1 as a candidate gene for the SPG15 locus, but our mutational analysis possibly excludes RTN1 as causative gene.