Somer Lilach, Shmulman Oshrit, Dror Tali, Hashmueli Sharon, Kashi Yechezkel
Faculty of Food Engineering and Biotechnology, Technion, Haifa, Israel.
Cell Stress Chaperones. 2002 Jan;7(1):47-54. doi: 10.1379/1466-1268(2002)007<0047:teccia>2.0.co;2.
The eukaryotic Hsp60 cytoplasmic chaperonin CCT (chaperonin containing the T-complex polypeptide-1) is essential for growth in budding yeast, and mutations in individual CCT subunits have been shown to affect assembly of tubulin and actin. The present research focused mainly on the expression of the CCT subunits, CCTalpha and CCTbeta, in yeast (Saccharomyces cerevisiae). Previous studies showed that, unlike most other chaperones, CCT in yeast does not undergo induction following heat shock. In this study, messenger ribonucleic acid (mRNA) and protein levels of CCT subunits following exposure to low temperatures, were examined. The Northern blot analysis indicated a 3- to 4-fold increase in mRNA levels of CCTalpha and CCTbeta genes after cold shock at 4 degrees C. Interestingly, Western blot analysis showed that cold shock induces an increase in the CCTalpha protein, which is expressed at 10 degrees C, but not at 4 degrees C. Transfer of 4 degrees C cold-shocked cells to 10 degrees C induced a 5-fold increase in the CCTalpha protein level. By means of fluorescent immunostaining and confocal microscopy, we found CCTalpha to be localized in the cortex and the cell cytoplasm of S. cerevisiae. Localization of CCTalpha was not affected at low temperatures. Co-localization of CCT and filaments of actin and tubulin was not observed by microscopy. The induction pattern of the CCTalpha protein suggests that expression of the chaperonin may be primarily important during the recovery from low temperatures and the transition to growth at higher temperatures, as found for other Hsps during the recovery phase from heat shock.
真核生物的热休克蛋白60细胞质伴侣蛋白CCT（含T复合物多肽-1的伴侣蛋白）对于芽殖酵母的生长至关重要，并且已表明单个CCT亚基的突变会影响微管蛋白和肌动蛋白的组装。本研究主要聚焦于酵母（酿酒酵母）中CCT亚基CCTα和CCTβ的表达。先前的研究表明，与大多数其他伴侣蛋白不同，酵母中的CCT在热休克后不会被诱导。在本研究中，检测了暴露于低温后CCT亚基的信使核糖核酸（mRNA）和蛋白质水平。Northern印迹分析表明，在4℃冷休克后，CCTα和CCTβ基因的mRNA水平增加了3至4倍。有趣的是，蛋白质印迹分析表明，冷休克诱导了CCTα蛋白的增加，该蛋白在10℃时表达，但在4℃时不表达。将4℃冷休克的细胞转移至10℃会导致CCTα蛋白水平增加5倍。通过荧光免疫染色和共聚焦显微镜，我们发现CCTα定位于酿酒酵母的皮质和细胞质中。CCTα的定位在低温下不受影响。显微镜检查未观察到CCT与肌动蛋白和微管蛋白丝的共定位。CCTα蛋白的诱导模式表明，伴侣蛋白的表达在从低温恢复以及向较高温度生长的转变过程中可能至关重要，正如在热休克恢复阶段其他热休克蛋白的情况一样。
