Mannan Ashraf U, Boehm Johann, Sauter Simone M, Rauber Anne, Byrne Paula C, Neesen Juergen, Engel Wolfgang
Institute of Human Genetics, University of Goettingen, Heinrich-Dueker-Weg 12, Goettingen 37073, Germany.
Neurogenetics. 2006 May;7(2):93-103. doi: 10.1007/s10048-006-0034-4. Epub 2006 Apr 7.
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.
痉挛素是一种属于AAA蛋白家族的ATP酶，在常染色体显性遗传性痉挛性截瘫（HSP）中最常发生突变。痉挛素是一种多面性蛋白，在细胞活动中具有多种作用，主要参与微管动力学。为了进一步深入了解痉挛素的分子功能，我们使用酵母双杂交方法来鉴定痉挛素的新型相互作用伙伴。以痉挛素作为诱饵，我们鉴定出网织蛋白1（RTN1）和网织蛋白3（RTN3）为潜在的与痉挛素相互作用的蛋白。RTN1和RTN3属于网织蛋白（RTN）基因家族，主要在内质网中表达。此外，已知RTN1在囊泡运输过程中发挥作用。通过体外和体内免疫沉淀实验，我们能够证明RTN1与痉挛素特异性相互作用。使用免疫染色和表位标记蛋白的过表达进行的细胞内分布研究显示，痉挛素和RTN1在细胞质中的离散囊泡中明显共定位。痉挛素通过其包含微管相互作用和运输结构域的N末端区域介导其与RTN1的相互作用。有趣的是，与RTN1共表达可挽救截短的痉挛素蛋白异常的细胞内分布，这突出了这种相互作用的生理意义。我们的发现强化了细胞内囊泡运输过程中断可能导致HSP的假说。有趣的是，RTN1定位于14q23.1，而SPG15基因座也定位在此处。因此，我们将RTN1视为SPG15基因座的候选基因，但我们的突变分析可能排除了RTN1作为致病基因。
