Kalish J E, Keller G A, Morrell J C, Mihalik S J, Smith B, Cregg J M, Gould S J
Department of Biological Chemistry, The John Hopkins University School of Medicine, Baltimore, MD 21205, USA.
EMBO J. 1996 Jul 1;15(13):3275-85.
Fluorescent peroxisomal probes were developed by fusing green fluorescent protein (GFP) to the matrix peroxisomal targeting signals PTS1 and PTS2, as well as to an integral peroxisomal membrane protein (IPMP). These proteins were used to identify and characterize novel peroxisome assembly (pas) mutants in the yeast Pichia pastoris. Mutant cells lacking the PAS10 gene mislocalized both PTS1-GFP and PTS2-GFP to the cytoplasm but did incorporate IPMP-GFP into peroxisome membranes. Similar distributions were observed for endogenous peroxisomal matrix and membrane proteins. While peroxisomes from translocation-competent pas mutants sediment in sucrose gradients at the density of normal peroxisomes, >98% of peroxisomes from pas10 cells migrated to a much lower density and had an extremely low ratio of matrix:membrane protein. These data indicate that Pas10p plays an important role in protein translocation across the peroxisome membrane. Consistent with this hypothesis, we find that Pas10p is an integral protein of the peroxisome membrane. In addition, Pas10p contains a cytoplasmically-oriented C3HC4 zinc binding domain that is essential for its biological activity.
通过将绿色荧光蛋白（GFP）与过氧化物酶体基质靶向信号PTS1和PTS2以及完整的过氧化物酶体膜蛋白（IPMP）融合，开发出了荧光过氧化物酶体探针。这些蛋白质被用于鉴定和表征酵母毕赤酵母中新型过氧化物酶体组装（pas）突变体。缺乏PAS10基因的突变细胞将PTS1-GFP和PTS2-GFP都错误定位于细胞质中，但确实将IPMP-GFP整合到了过氧化物酶体膜中。内源性过氧化物酶体基质和膜蛋白也观察到类似的分布。虽然来自具有转运能力的pas突变体的过氧化物酶体在蔗糖梯度中以正常过氧化物酶体的密度沉降，但来自pas10细胞的过氧化物酶体中>98%迁移到低得多的密度，并且基质：膜蛋白的比例极低。这些数据表明Pas10p在蛋白质穿过过氧化物酶体膜的转运中起重要作用。与这一假设一致，我们发现Pas10p是过氧化物酶体膜的整合蛋白。此外，Pas10p包含一个面向细胞质的C3HC4锌结合结构域，该结构域对其生物学活性至关重要。
