Identification of protein components in yeast mitochondrial nuclei.

1.我们从厌氧生长的细胞、呼吸缺陷细胞和呼吸抑制细胞中分离线粒体核。我们发现，与有氧培养细胞的线粒体核相比，这些线粒体核中67 kDa蛋白的含量显著降低.测定了67 kDa蛋白的N-末端氨基酸序列。该蛋白被证明是肉毒碱乙酰转移酶（CAT），定位于线粒体。抗CAT抗体的免疫学检测表明CAT与线粒体核团结合。用荧光显微镜观察了三角酵母中线粒体、线粒体核、微管和肌动蛋白的行为.我们证明，Abf2p，线粒体的主要DNA结合蛋白，是紧密相关的线粒体核蛋白免疫印迹和免疫荧光显微镜。SDS-DNA PAGE是检测Abf2p的有效方法.我们鉴定了能被YMN-1单克隆抗体识别的线粒体核YMN-1抗原蛋白为二氢硫辛酰转琥珀酰酶（dihydrolipoyl transsuccinylase，KE 2）。酶活性测定表明，α-酮戊二酸脱氢酶复合物（KGDC）与线粒体核在蔗糖浓度梯度下共沉淀.线粒体核蛋白组分通过二维（2D）电泳分离。我们测定了线粒体核内54 kDa、52 kDa、50 kDa、38 kDa和22 kDa蛋白质的N端氨基酸序列。

1. We isolated mitochondrial (mt) nuclei from anaerobically-grown cells, respiratory deficient cells and respiration-inhibited cells. We found that these mt-nuclei contained significantly reduced amount of 67 kDa protein, compared with mt-nuclei from aerobically-grown cells.2. The N-terminal amino acid sequence of the 67 kDa protein was determined. This protein was proved to be a carnitine acetyltransferase (CAT) that localized in mitochondria. Immunological assay using the antibody against CAT suggested association of CAT with mt-nuclei.3. Behavior of mitochondria, mt-nuclei, microtubules and actin were investigated by fluorescence microscopy in the triangular yeast, Trigonopsis variabilis.4. We demonstrated that Abf2p, a major DNA-binding protein of mitochondria, is tightly associated with mt-nuclei by Western blotting and immunofluorescence microscopy. We showed that SDS-DNA PAGE is an efficient for detection of Abf2p on gels.5. We identified the YMN-1 antigen protein of mt-nuclei, which is recognized by YMN-1 monoclonal antibody, as dihydrolipoyl transsuccinylase (KE2). Enzyme assay suggested that α-ketoglutarate dehydrogenase complex (KGDC) cosedimented with mt-nuclei in sucrose-density gradient.6. Protein components of mt-nuclei were separated by two-dimensional (2D) electrophoresis. We determined N-terminal amino acid sequence of 54 kDa, 52 kDa, 50 kDa, 38 kDa and 22 kDa proteins, respectively, which are involved in mt-nuclei.