Zhang J W, Lazarow P B
Department of Cell Biology and Anatomy, Mount Sinai School of Medicine, New York 10029, USA.
J Cell Biol. 1996 Feb;132(3):325-34. doi: 10.1083/jcb.132.3.325.
Peb1 is a peroxisome biogenesis mutant isolated in Saccharomyces cerevisiae that is selectively defective in the import of thiolase into peroxisomes but has a normal ability to package catalase, luciferase and acyl-CoA oxidase (Zhang, J. W., C. Luckey, and P. B. Lazarow. 1993. Mol. Biol. Cell. 4:1351-1359). Thiolase differs from these other peroxisomal proteins in that it is targeted by an NH2-terminal, 16-amino acid peroxisomal targeting sequence type 2 (PTS 2). This phenotype suggests that the PEB1 protein might function as a receptor for the PTS2. The PEB1 gene has been cloned by functional complementation. It encodes a 42,320-D, hydrophilic protein with no predicted transmembrane segment. It contains six WD repeats that comprise the entire protein except for the first 55 amino acids. Peb1p was tagged with hemagglutinin epitopes and determined to be exclusively within peroxisomes by digitonin permeabilization, immunofluorescence, protease protection and immuno-electron microscopy (Zhang, J. W., and P. B. Lazarow. 1995. J. Cell Biol. 129:65-80). Peb1p is identical to Pas7p (Marzioch, M., R. Erdmann, M. Veenhuis, and W.-H. Kunau. 1994. EMBO J. 13: 4908-4917). We have now tested whether Peb1p interacts with the PTS2 of thiolase. With the two-hybrid assay, we observed a strong interaction between Peb1p and thiolase that was abolished by deleting the first 16 amino acids of thiolase. An oligopeptide consisting of the first 16 amino acids of thiolase was sufficient for the affinity binding of Peb1p. Binding was reduced by the replacement of leucine with arginine at residue five, a change that is known to reduce thiolase targeting in vivo. Finally, a thiolase-Peb1p complex was isolated by immunoprecipitation. To investigate the topogenesis of Peb1p, its first 56-amino acid residues were fused in front of truncated thiolase lacking the NH2-terminal 16-amino acid PTS2. The fusion protein was expressed in a thiolase knockout strain. Equilibrium density centrifugation and immunofluorescence indicated that the fusion protein was located in peroxisomes. Deletion of residues 6-55 from native Peb1p resulted in a cytosolic location and the loss of function. Thus the NH2-terminal 56-amino acid residues of Peb1p are necessary and sufficient for peroxisomal targeting. Peb1p is found in peroxisomes whether thiolase is expressed or not. These results suggest that Peb1p (Pas7p) is an intraperoxisomal receptor for the type 2 peroxisomal targeting signal.
Peb1是在酿酒酵母中分离出的一种过氧化物酶体生物发生突变体，它在硫解酶导入过氧化物酶体的过程中存在选择性缺陷，但在包装过氧化氢酶、荧光素酶和酰基辅酶A氧化酶方面能力正常（Zhang, J. W., C. Luckey, and P. B. Lazarow. 1993. Mol. Biol. Cell. 4:1351 - 1359）。硫解酶与这些其他过氧化物酶体蛋白的不同之处在于，它由一个氨基末端的16个氨基酸的2型过氧化物酶体靶向序列（PTS 2）靶向。这种表型表明PEB1蛋白可能作为PTS2的受体发挥作用。PEB1基因已通过功能互补进行克隆。它编码一种42320道尔顿的亲水性蛋白，没有预测的跨膜区段。它包含六个WD重复序列，除了前55个氨基酸外，构成了整个蛋白质。通过用血细胞凝集素表位标记Peb1p，并通过洋地黄皂苷通透化、免疫荧光、蛋白酶保护和免疫电子显微镜确定其仅存在于过氧化物酶体中（Zhang, J. W., and P. B. Lazarow. 1995. J. Cell Biol. 129:65 - 80）。Peb1p与Pas7p相同（Marzioch, M., R. Erdmann, M. Veenhuis, and W.-H. Kunau. 1994. EMBO J. 13: 4908 - 4917）。我们现在测试了Peb1p是否与硫解酶的PTS2相互作用。通过双杂交试验，我们观察到Peb1p与硫解酶之间有强烈的相互作用，这种相互作用在删除硫解酶的前16个氨基酸后被消除。由硫解酶的前16个氨基酸组成的寡肽足以实现Peb1p的亲和结合。在第5位残基处用精氨酸取代亮氨酸会降低结合，已知这种变化会减少硫解酶在体内的靶向作用。最后，通过免疫沉淀分离出硫解酶 - Peb1p复合物。为了研究Peb1p的拓扑发生，将其前56个氨基酸残基融合到缺乏氨基末端16个氨基酸PTS2的截短硫解酶前面。融合蛋白在硫解酶敲除菌株中表达。平衡密度离心和免疫荧光表明融合蛋白位于过氧化物酶体中。从天然Peb1p中删除第6 - 55位残基导致其位于细胞质中且功能丧失。因此，Peb1p的氨基末端56个氨基酸残基对于过氧化物酶体靶向是必要且充分的。无论硫解酶是否表达，Peb1p都存在于过氧化物酶体中。这些结果表明Peb1p（Pas7p）是2型过氧化物酶体靶向信号的过氧化物酶体内受体。
