Hillebrand Merle, Verrier Sophie E, Ohlenbusch Andreas, Schäfer Annika, Söling Hans-Dieter, Wouters Fred S, Gärtner Jutta
Department of Pediatrics and Pediatric Neurology, Georg August University, Faculty of Medicine, Robert-Koch-Strasse 40, 37075 Göttingen, Germany.
Department of Neurobiology, Max Planck Institute of Biophysical Chemistry, Am Fassberg 11, 37077 Göttingen, Germany.
J Biol Chem. 2007 Sep 14;282(37):26997-27005. doi: 10.1074/jbc.M702122200. Epub 2007 Jul 3.
The adrenoleukodystrophy protein (ALDP) and the 70-kDa peroxisomal membrane protein (PMP70) are half-ATP-binding cassette (ABC) transporters in the mammalian peroxisome membrane. Mutations in the gene encoding ALDP result in a devastating neurodegenerative disorder, X-linked adrenoleukodystrophy (X-ALD) that is associated with elevated levels of very long chain fatty acids because of impaired peroxisomal beta-oxidation. The interactions of peroxisomal ABC transporters, their role in the peroxisomal membrane, and their functions in disease pathogenesis are poorly understood. Studies on ABC transporters revealed that half-transporters have to dimerize to gain functionality. So far, conflicting observations are described for ALDP. By the use of in vitro methods (yeast two-hybrid and immunoprecipitation assays) on the one hand, it was shown that ALDP can form homodimers as well as heterodimers with PMP70 and ALDR, while on the other hand, it was demonstrated that ALDP and PMP70 exclusively homodimerize. To circumvent the problems of artificial interactions due to biochemical sample preparation in vitro, we investigated protein-protein interaction of ALDP in its physiological environment by FRET microscopy in intact living cells. The statistical relevance of FRET data was determined in two different ways using probability distribution shift analysis and Kolmogorov-Smirnov statistics. We demonstrate in vivo that ALDP and PMP70 form homodimers as well as ALDP/PMP70 heterodimers where ALDP homodimers predominate. Using C-terminal deletion constructs of ALDP, we demonstrate that the last 87 C-terminal amino acids harbor the most important protein domain mediating these interactions, and that the N-terminal transmembrane region of ALDP has an additional stabilization effect on ALDP homodimers. Loss of ALDP homo- or heterodimerization is highly relevant for understanding the disease mechanisms of X-ALD.
肾上腺脑白质营养不良蛋白（ALDP）和70 kDa过氧化物酶体膜蛋白（PMP70）是哺乳动物过氧化物酶体膜中的半ATP结合盒（ABC）转运蛋白。编码ALDP的基因突变会导致一种毁灭性的神经退行性疾病——X连锁肾上腺脑白质营养不良（X-ALD），由于过氧化物酶体β氧化受损，该病与超长链脂肪酸水平升高有关。过氧化物酶体ABC转运蛋白的相互作用、它们在过氧化物酶体膜中的作用以及它们在疾病发病机制中的功能目前尚不清楚。对ABC转运蛋白的研究表明，半转运蛋白必须二聚化才能获得功能。到目前为止，关于ALDP的观察结果相互矛盾。一方面，通过体外方法（酵母双杂交和免疫沉淀试验）表明，ALDP可以与PMP70和ALDR形成同二聚体以及异二聚体，而另一方面，又证明ALDP和PMP70仅形成同二聚体。为了避免体外生化样品制备导致的人工相互作用问题，我们通过完整活细胞中的荧光共振能量转移（FRET）显微镜研究了ALDP在其生理环境中的蛋白质-蛋白质相互作用。使用概率分布转移分析和柯尔莫哥洛夫-斯米尔诺夫统计两种不同方法确定了FRET数据的统计相关性。我们在体内证明，ALDP和PMP70形成同二聚体以及ALDP/PMP70异二聚体，其中ALDP同二聚体占主导。使用ALDP的C端缺失构建体，我们证明C端最后87个氨基酸包含介导这些相互作用的最重要蛋白质结构域，并且ALDP的N端跨膜区域对ALDP同二聚体有额外的稳定作用。ALDP同二聚化或异二聚化的丧失对于理解X-ALD的疾病机制高度相关。
