Mooney D T, Pilgrim D B, Young E T
Department of Biochemistry, University of Washington, Seattle 98195.
Mol Cell Biol. 1990 Jun;10(6):2801-8. doi: 10.1128/mcb.10.6.2801-2808.1990.
Point mutations in the presequence of the mitochondrial alcohol dehydrogerase isoenzyme (ADH III) have been shown to affect either the import of the precursor protein into yeast mitochondria in vivo or its processing within the organelle. In the present work, the behavior of these mutants during in vitro import into isolated mitochondria was investigated. All point mutants tested were imported with a slower initial rate than that of the wild-type precursor. This defect was corrected when the precursors were treated with urea prior to import. Once imported, the extent of processing to the mature form of mutant precursors varied greatly and correlated well with the defects observed in vivo. This result was not affected by prior urea treatment. When matrix extracts enriched for the processing protease were used, this defect was shown to be due to failure of the protease to efficiently recognize or cleave the presequence, rather than to a lack of access to the precursor. The rate of import of two ADH III precursors bearing internal deletions in the leader sequence was similar to those of the point mutants, whereas a deletion leading to the removal of the 15 amino-terminal amino acids was poorly imported. The mature amino terminus of wild-type ADH III was determined to be Gln-25. Mutant m01 (Ser-26 to Phe), which reduced the efficiency of cleavage in vitro by 80%, was cleaved at the correct site.
线粒体乙醇脱氢酶同工酶（ADH III）前序列中的点突变已被证明会影响前体蛋白在体内导入酵母线粒体的过程，或其在细胞器内的加工过程。在本研究中，对这些突变体在体外导入分离线粒体过程中的行为进行了研究。所有测试的点突变体导入的初始速率均比野生型前体慢。在导入前用尿素处理前体时，这种缺陷得到了纠正。一旦导入，突变体前体加工成成熟形式的程度差异很大，并且与在体内观察到的缺陷密切相关。这一结果不受尿素预处理的影响。当使用富含加工蛋白酶的基质提取物时，这种缺陷被证明是由于蛋白酶未能有效识别或切割前序列，而不是由于无法接触到前体。两种在引导序列中带有内部缺失的ADH III前体的导入速率与点突变体相似，而导致去除15个氨基末端氨基酸的缺失突变体导入效率很低。野生型ADH III的成熟氨基末端被确定为Gln-25。突变体m01（Ser-26突变为Phe）在体外切割效率降低了80%，但仍在正确位点被切割。
