Fenton W A, Hack A M, Kraus J P, Rosenberg L E
Proc Natl Acad Sci U S A. 1987 Mar;84(5):1421-4. doi: 10.1073/pnas.84.5.1421.
Methylmalonyl-CoA mutase (2-methylmalonyl-CoA CoA-carbonylmutase, EC 5.4.99.2) is a mitochondrial enzyme whose deficiency in man leads to several biochemically and clinically heterogenous++ forms of methylmalonic acidemia. Intact fibroblasts from 21 patients with mutase apoenzyme deficiency have been pulse-labeled with [3H]leucine or [35S]methionine to determine how amounts of newly synthesized mutase recovered from these cells by immunoprecipitation compare with the amounts of steady-state crossreacting material previously determined. Ten lines (3 mut-, 7 mut 0 ), previously shown to have detectable steady-state crossreacting material, had amounts of newly synthesized mutase that varied from similar (7 lines) to considerably greater than (3 lines) the steady-state amounts. Of 11 lines that had no detectable steady-state crossreacting material, 6 had no detectable newly synthesized mutase, and 5 had amounts of mutase ranging from just detectable to almost half that of control. This result suggests that, at least for this latter group, one effect of the mutation in the mutase gene is to reduce the stability of the mutase protein. We examined fibroblasts from 48 patients with mutase apoenzyme deficiency to determine the sizes of the mature mutase subunit and the mutase precursor accumulated in the presence of the mitochondrial transport inhibitor rhodamine 6G. Of the 38 lines that had detectable newly synthesized mutase, only 2, lines 437 and 552, showed a pattern different from that generated by the normal precursor and mature subunits. Line 437 had two immunoprecipitable precursor proteins in the presence of rhodamine, each of which appeared to be transported and processed in the cells to produce two distinct mature proteins. Line 552 also had two anti-mutase reactive proteins in the presence of rhodamine, but each was smaller than the normal mature subunit and neither appeared to be proteolytically processed. The defect in line 552 is almost certainly an amino-terminal deletion that removes the leader peptide necessary for proper uptake and cleavage of the mutase precursor; this represents a clear example of a natural human mutation that interferes with mitochondrial transport of a protein.
甲基丙二酰辅酶A变位酶（2-甲基丙二酰辅酶A 辅酶A-羰基变位酶，EC 5.4.99.2）是一种线粒体酶，人类缺乏该酶会导致几种生化和临床异质性的甲基丙二酸血症形式。对21例变位酶脱辅基酶缺乏患者的完整成纤维细胞用[3H]亮氨酸或[35S]甲硫氨酸进行脉冲标记，以确定通过免疫沉淀从这些细胞中回收的新合成变位酶的量与先前测定的稳态交叉反应物质的量相比如何。10个细胞系（3个mut-，7个mut 0），先前已证明有可检测到的稳态交叉反应物质，其新合成变位酶的量从相似（7个细胞系）到远大于（3个细胞系）稳态量不等。在11个未检测到稳态交叉反应物质的细胞系中，6个未检测到新合成的变位酶，5个细胞系的变位酶量从刚可检测到到几乎是对照的一半不等。这一结果表明，至少对于后一组细胞系，变位酶基因突变的一个作用是降低变位酶蛋白的稳定性。我们检查了48例变位酶脱辅基酶缺乏患者的成纤维细胞，以确定在存在线粒体转运抑制剂罗丹明6G 的情况下积累的成熟变位酶亚基和变位酶前体的大小。在38个检测到新合成变位酶的细胞系中，只有2个细胞系，即437和552细胞系，显示出与正常前体和成熟亚基产生的模式不同。在罗丹明存在的情况下，437细胞系有两种可免疫沉淀的前体蛋白，每种蛋白似乎都在细胞中被转运和加工以产生两种不同的成熟蛋白。在罗丹明存在的情况下，552细胞系也有两种抗变位酶反应性蛋白，但每种蛋白都比正常成熟亚基小，且似乎都未经过蛋白水解加工。552细胞系的缺陷几乎肯定是氨基末端缺失，该缺失去除了变位酶前体正确摄取和切割所需的前导肽；这代表了一个明显的人类自然突变干扰蛋白质线粒体转运的例子。
