Keng T, Alani E, Guarente L
Mol Cell Biol. 1986 Feb;6(2):355-64. doi: 10.1128/mcb.6.2.355-364.1986.
delta-Aminolevulinate synthase, the first enzyme in the heme biosynthetic pathway, is encoded by the nuclear gene HEM1. The enzyme is synthesized as a precursor in the cytoplasm and imported into the matrix of the mitochondria, where it is processed to its mature form. Fusions of beta-galactosidase to various lengths of amino-terminal fragments of delta-aminolevulinate synthase were constructed and transformed into yeast cells. The subcellular location of the fusion proteins was determined by organelle fractionation. Fusion proteins were found to be associated with the mitochondria. Protease protection experiments involving the use of intact mitochondria or mitoplasts localized the fusion proteins to the mitochondrial matrix. This observation was confirmed by fractionation of the mitochondrial compartments and specific activity measurements of beta-galactosidase activity. The shortest fusion protein contains nine amino acid residues of delta-aminolevulinate synthase, indicating that nine amino-terminal residues are sufficient to localize beta-galactosidase to the mitochondrial matrix. The amino acid sequence deduced from the DNA sequence of HEM1 showed that the amino-terminal region of delta-aminolevulinate synthase was largely hydrophobic, with a few basic residues interspersed.
δ-氨基-γ-酮戊酸合酶是血红素生物合成途径中的第一个酶，由核基因HEM1编码。该酶在细胞质中以前体形式合成，然后导入线粒体基质，在那里加工成成熟形式。构建了β-半乳糖苷酶与δ-氨基-γ-酮戊酸合酶不同长度氨基末端片段的融合体，并将其转化到酵母细胞中。通过细胞器分级分离确定融合蛋白的亚细胞定位。发现融合蛋白与线粒体相关。使用完整线粒体或线粒体膜间隙进行的蛋白酶保护实验将融合蛋白定位到线粒体基质。通过线粒体区室分级分离和β-半乳糖苷酶活性的比活性测量证实了这一观察结果。最短的融合蛋白包含δ-氨基-γ-酮戊酸合酶的九个氨基酸残基，表明九个氨基末端残基足以将β-半乳糖苷酶定位到线粒体基质。从HEM1的DNA序列推导的氨基酸序列表明，δ-氨基-γ-酮戊酸合酶的氨基末端区域主要是疏水的，散布着一些碱性残基。
