Brickner D G, Olsen L J
Department of Biology, University of Michigan, Ann Arbor 48109-1048, USA.
Plant Physiol. 1998 Jan;116(1):309-17. doi: 10.1104/pp.116.1.309.
All peroxisomal proteins are nuclear encoded, synthesized on free cytosolic ribosomes, and posttranslationally targeted to the organelle. We have used an in vitro assay to reconstitute protein import into pumpkin (Cucurbita pepo) glyoxysomes, a class of peroxisome found in the cotyledons of oilseed plants, to study the mechanisms involved in protein transport across peroxisome membranes. Results indicate that ATP hydrolysis is required for protein import into peroxisomes; nonhydrolyzable analogs of ATP could not substitute for this requirement. Nucleotide competition studies suggest that there may be a nucleotide binding site on a component of the translocation machinery. Peroxisomal protein import also was supported by GTP hydrolysis. Nonhydrolyzable analogs of GTP did not substitute in this process. Experiments to determine the cation specificity of the nucleotide requirement show that the Mg2+ salt was preferred over other divalent and monovalent cations. The role of a putative protonmotive force across the peroxisomal membrane was also examined. Although low concentrations of ionophores had no effect on protein import, relatively high concentrations of all ionophores tested consistently reduced the level of protein import by approximately 50%. This result suggests that a protonmotive force is not absolutely required for peroxisomal protein import.
所有过氧化物酶体蛋白都是由细胞核编码的，在游离的胞质核糖体上合成，并在翻译后靶向运输到该细胞器。我们利用一种体外测定法来重建蛋白质导入南瓜（西葫芦）乙醛酸循环体的过程，乙醛酸循环体是在油料植物子叶中发现的一类过氧化物酶体，以此研究蛋白质跨过氧化物体膜运输所涉及的机制。结果表明，蛋白质导入过氧化物酶体需要ATP水解；ATP的不可水解类似物不能替代这一需求。核苷酸竞争研究表明，转运机制的一个组分上可能存在一个核苷酸结合位点。GTP水解也支持过氧化物酶体蛋白的导入。GTP的不可水解类似物在此过程中不能替代。确定核苷酸需求的阳离子特异性的实验表明，Mg2+盐比其他二价和一价阳离子更受青睐。还研究了过氧化物酶体膜两侧假定的质子动力势的作用。虽然低浓度的离子载体对蛋白质导入没有影响，但所测试的所有离子载体的相对高浓度一致地使蛋白质导入水平降低了约50%。这一结果表明，过氧化物酶体蛋白导入并非绝对需要质子动力势。
