Morgan Rhian R, Errington Rachel, Elder George H
Department of Medical Biochemistry and Immunology, University of Wales College of Medicine, Heath Park, Cardiff CF14 4XN, UK.
Biochem J. 2004 Jan 15;377(Pt 2):281-7. doi: 10.1042/BJ20030978.
Protoporphyrinogen oxidase (PPOX; EC 1.3.3.4), the penultimate enzyme of haem biosynthesis, is a nucleus-encoded flavoprotein strongly associated with the outer surface of the inner mitochondrial membrane. It is attached to this membrane by an unknown mechanism that appears not to involve a membrane-spanning domain. The pathway for its import to mitochondria and insertion into the inner membrane has not been established. We have fused human PPOXs containing N-terminal deletions, C-terminal deletions or missense mutations to yellow fluorescent protein (YFP) and have used these constructs to investigate the mitochondrial import of PPOX in human cells. We show that all the information required for efficient import is contained within the first 250 amino acid residues of human PPOX and that targeting to mitochondria is prevented by fusion of YFP to the N-terminus. Deletion of between 151 and 175 residues from the N-terminus is required to abolish import, whereas shorter deletions impair its efficiency. Fully efficient targeting appears to require both a major targeting signal, the whole or part of which is contained between residues 151 and 175, and which may be involved in anchoring to the inner mitochondrial membrane, together with interaction between this region and a sequence(s) within the first 150 residues. These features suggest that the mechanism for import of human PPOX to mitochondria differs from those identified for the translocation of nucleus-encoded, membrane-spanning, inner membrane proteins. In addition, a missense mutation outside this region (Val(335)-->Gly) prevented targeting to mitochondria and delayed the appearance of YFP fluorescence. This mutation appeared to prevent import by a direct effect on protein folding rather than by altering a sequence required for targeting. It may lead to sequestration of the PPOX-YFP construct in an unfolded conformation, followed by proteolytic degradation, possibly through enhanced binding to a cytosolic chaperone protein.
原卟啉原氧化酶（PPOX；EC 1.3.3.4）是血红素生物合成的倒数第二个酶，是一种核编码黄素蛋白，与线粒体内膜外表面紧密相关。它通过一种未知机制附着于该膜上，这种机制似乎不涉及跨膜结构域。其导入线粒体并插入内膜的途径尚未明确。我们将含有N端缺失、C端缺失或错义突变的人PPOX与黄色荧光蛋白（YFP）融合，并利用这些构建体研究人细胞中PPOX的线粒体导入。我们发现，高效导入所需的所有信息都包含在人PPOX的前250个氨基酸残基内，并且YFP与N端融合会阻止其靶向线粒体。从N端删除151至175个残基才能消除导入，而较短的删除会损害其效率。完全有效的靶向似乎需要一个主要靶向信号，其全部或部分包含在151至175位残基之间，可能参与锚定到线粒体内膜，同时该区域与前150个残基内的一个或多个序列相互作用。这些特征表明，人PPOX导入线粒体的机制与已确定的核编码跨膜内膜蛋白的转运机制不同。此外，该区域外的一个错义突变（Val(335)-->Gly）阻止了靶向线粒体并延迟了YFP荧光的出现。这种突变似乎通过直接影响蛋白质折叠而不是通过改变靶向所需的序列来阻止导入。它可能导致PPOX - YFP构建体以未折叠构象被隔离，随后被蛋白水解降解，可能是通过增强与胞质伴侣蛋白的结合。
