An oligomeric protein is imported into peroxisomes in vivo.
An oligomeric protein is imported into peroxisomes in vivo.
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DOI：
10.1083/jcb.127.5.1245
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发表时间：
1994-12
期刊：
影响因子：
--
通讯作者：
Goodman JM
中科院分区：
文献类型：
--
作者：
McNew JA;Goodman JM
过氧化物酶体蛋白从细胞质易位到基质中的机制在很大程度上是未知的。我们一直在酵母中研究这个问题。我们表明，过氧化物酶体靶向序列SKL或AKL，有或没有9个甘氨酸（G9）的间隔区，是足够的目标氯霉素乙酰转移酶（CAT）的过氧化物酶体的酿酒酵母在体内。CAT的成熟形式是同源三聚体，并且发现CAT的完全三聚化在合成的几分钟内发生。与此相反，进口，通过免疫电镜和细胞器分馏测量，发生在几个小时。为了确认发生了预组装的CAT三聚体的输入，我们共表达了CAT-G9-AKL与缺乏过氧化物酶体靶向序列但含有血凝素衍生的表位标签（HA-CAT）的CAT。我们发现HA-CAT不被导入，除非它与CAT-G9-AKL共表达。这两种蛋白质释放的细胞器在温和的条件下（pH 8.5），释放其他基质蛋白，表明进口已经发生。这些结果强烈地表明HA-CAT是作为与CAT-G9-AKL的异源三聚体输入的。寡聚体输入的过程也发生在动物细胞中。当HA-CAT与CAT-G9-AKL在CV-1细胞中共表达时，HA-CAT与过氧化物酶体共定位，但当单独表达时在细胞质中。目前尚不清楚是否进口的球状蛋白进入过氧化物酶体发生通过过氧化物酶体膜孔或涉及膜内化。这两种可能性进行了讨论。
The mechanism of translocation of peroxisomal proteins from the cytoplasm into the matrix is largely unknown. We have been studying this problem in yeast. We show that the peroxisomal targeting sequences SKL or AKL, with or without a spacer of nine glycines (G9), are sufficient to target chloramphenicol acetyltransferase (CAT) to peroxisomes of Saccharomyces cerevisiae in vivo. The mature form of CAT is a homotrimer, and complete trimerization of CAT was found to occur within a few minutes of synthesis. In contrast, import, measured by immunoelectron microscopy and organellar fractionation, occurred over several hours. To confirm that import of preassembled CAT trimers was occurring, we co-expressed CAT-G9-AKL with CAT lacking a peroxisomal targeting sequence but containing a hemagglutinin-derived epitope tag (HA-CAT). We found that HA-CAT was not imported unless it was co- expressed with CAT-G9-AKL. Both proteins were released from the organelles under mild conditions (pH 8.5) that released other matrix proteins, indicating that import had occurred. These results strongly suggested that HA-CAT was imported as a heterotrimer with CAT-G9-AKL. The process of oligomeric import also occurs in animal cells. When HA- CAT was co-expressed with CAT-G9-AKL in CV-1 cells, HA-CAT co-localized with peroxisomes but was cytoplasmic when expressed alone. It is not clear whether the import of globular proteins into peroxisomes occurs through peroxisomal membrane pores or involves membrane internalization. Both possibilities are discussed.