Graduate School of Life Science, University of Hyogo, Kouto Ako-gun, Japan.
J Biochem. 2010 Apr;147(4):581-90. doi: 10.1093/jb/mvp205. Epub 2009 Dec 10.
Most membrane proteins are recognized by a signal recognition particle and are cotranslationally targeted to the endoplasmic reticulum (ER) membrane, whereas almost all peroxisomal membrane proteins are posttranslationally targeted to the destination. Here we examined organelle-targeting properties of the N-terminal portions of the peroxisomal isoform of the ABC transporter PMP70 (ABCD3) using enhanced green fluorescent protein (EGFP) fusion. When the N-terminal 80 amino acid residue (N80)-segment preceding transmembrane segment (TM) 1 was deleted and the TM1-TM2 region was fused to EGFP, the TM1 segment induced ER-targeting and integration in COS cells. When the N80-segment was fused to EGFP, the fusion protein was targeted to the outer mitochondrial membrane. When both the N80-segment and the following TM1-TM2 region were present, the fusion located exclusively to the peroxisome. The full-length PMP70 molecule was clearly located in the ER in the absence of the N80-segment, even when multiple peroxisome-targeting signals were retained. We concluded that the TM1 segment possesses a sufficient ER-targeting function and that the N80-segment is critical for suppressing the ER-targeting function to allow the TM1-TM2 region to localize to the peroxisome. Cooperation of the organelle-targeting signals enables PMP70 to correctly target to peroxisomal membranes.
大多数膜蛋白都被信号识别颗粒识别，并被共翻译靶向内质网 (ER) 膜，而几乎所有过氧化物酶体膜蛋白都是翻译后靶向目标的。在这里，我们使用增强型绿色荧光蛋白 (EGFP) 融合研究了过氧化物酶体 ABC 转运蛋白 PMP70（ABCD3）的过氧化物酶体同工型的 N 端部分的细胞器靶向特性。当删除跨膜片段 (TM) 1 之前的 N 端 80 个氨基酸残基 (N80)-片段并将 TM1-TM2 区域融合到 EGFP 时，TM1 片段诱导 COS 细胞中的 ER 靶向和整合。当 N80-片段融合到 EGFP 时，融合蛋白被靶向到外线粒体膜。当 N80-片段和随后的 TM1-TM2 区域都存在时，融合蛋白仅定位在过氧化物酶体中。在不存在 N80-片段的情况下，全长 PMP70 分子显然位于 ER 中，即使保留了多个过氧化物酶体靶向信号。我们得出结论，TM1 片段具有足够的 ER 靶向功能，而 N80-片段对于抑制 ER 靶向功能以使 TM1-TM2 区域定位到过氧化物酶体至关重要。细胞器靶向信号的合作使 PMP70 能够正确靶向过氧化物酶体膜。
