Okuda A, Ohtsu M, Kimura G
Department of Virology, Medical Institute of Bioregulation, Kyushu University, Fukuoka, Japan.
Cell Struct Funct. 1999 Dec;24(6):443-9. doi: 10.1247/csf.24.443.
A temperature-sensitive mutant of 3Y1, 3Y1tsD123, reversibly arrested in G1 phase of cell cycle at the restrictive temperature of 39.8 degrees C, shows a single amino acid exchange in the D123 protein. In this study, we found that the D123 protein level in 3Y1tsD123, which was 1/8 of that in 3Y1 compared at the permissive temperature of 33.9 degrees C, lowered to 1/4 after a shift to the restrictive temperature. During inhibition of protein synthesis with cycloheximide, the D123 protein level in 3Y1tsD123 decreased markedly depending on the incubation temperature, compared with that in 3Y1, indicating that the lowered levels of D123 protein in 3Y1tsD123 are due to its degradation. Unexpectedly, 2 stably temperature-resistant clones were isolated after transfection of SV-3Y1tsD123 (SV40-transformed 3Y1tsD123, which shows cell death instead of G1 arrest at the restrictive temperature) with the cDNA of the mutant-type (3Y1tsD123-derived) D123 protein. The D123 protein in both clones degraded extensively at both temperatures, suggesting that the overexpression of the mutant-type D123 protein exceeds its degradation. Both temperature-resistant clones contained higher levels of D123 protein at the restrictive temperature than did SV-3Y1tsD123 at the permissive temperature. We concluded that the lowered D123 protein level at the restrictive temperature induces the temperature-sensitive characteristics of 3Y1tsD123 and SV-3Y1tsD123.
3Y1的一个温度敏感突变体3Y1tsD123在39.8摄氏度的限制温度下可逆地停滞在细胞周期的G1期，其D123蛋白存在一个氨基酸交换。在本研究中，我们发现，在33.9摄氏度的允许温度下比较时，3Y1tsD123中的D123蛋白水平是3Y1中的1/8，而在转移到限制温度后降低到了1/4。在用环己酰亚胺抑制蛋白质合成期间，与3Y1相比，3Y1tsD123中的D123蛋白水平根据孵育温度显著降低，这表明3Y1tsD123中D123蛋白水平的降低是由于其降解。出乎意料的是，在用突变型（源自3Y1tsD123）D123蛋白的cDNA转染SV - 3Y1tsD123（SV40转化的3Y1tsD123，在限制温度下表现为细胞死亡而非G1期停滞）后，分离出了2个稳定的耐温克隆。两个克隆中的D123蛋白在两个温度下均大量降解，这表明突变型D123蛋白的过表达超过了其降解。在限制温度下，两个耐温克隆中的D123蛋白水平均高于SV - 3Y1tsD123在允许温度下的水平。我们得出结论，在限制温度下降低的D123蛋白水平诱导了3Y1tsD123和SV - 3Y1tsD123的温度敏感特性。
