Microinjection of ubiquitin: changes in protein degradation in HeLa cells subjected to heat-shock.
Microinjection of ubiquitin: changes in protein degradation in HeLa cells subjected to heat-shock.
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DOI：
10.1083/jcb.104.3.547
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发表时间：
1987-03
影响因子：
7.8
通讯作者：
RECHSTEINER, M
中科院分区：
文献类型：
--
作者：
CARLSON, N;ROGERS, S;RECHSTEINER, M
用~(125)I-Bolton-Hunter试剂放射性标记泛素，通过红细胞介导的微量注射将泛素导入HeLa细胞。然后将注射的细胞在45℃下孵育5分钟(可逆热休克)或30分钟(致死性热休克)。在任一治疗后，泛素结合物的水平都发生了戏剧性的变化。在正常培养条件下，注射的泛素中约有10%与组蛋白相连，40%存在于分子量大于25000的结合物中，其余的是未结合的。热休克后，游离泛素库和组蛋白-泛素结合物水平迅速下降，以高相对分子质量的结合物为主。大的结合物的形成不需要蛋白质的合成；当用双向电泳法分析时，主要的结合物在热应激前后没有与热休克蛋白共迁移。在热休克的HeLa细胞中，伴随着游离泛素的丧失，内源性蛋白、注入的血红蛋白、BSA和泛素的降解减少。可逆热休克后，蛋白水解率下降不大，37℃孵育时，蛋白水解率和游离泛素库的大小均恢复到对照水平。然而，致命的热休克细胞比天然血红蛋白和在其内部形成的泛素-珠蛋白结合物更快地降解变性的血红蛋白。因此，热休克后蛋白质的稳定不能归因于泛素结合的丧失或不能降解与泛素形成结合的蛋白质。
Ubiquitin was radiolabeled by reaction with 125I-Bolton-Hunter reagent and introduced into HeLa cells using erythrocyte-mediated microinjection. The injected cells were then incubated at 45 degrees C for 5 min (reversible heat-shock) or for 30 min (lethal heat-shock). After either treatment, there were dramatic changes in the levels of ubiquitin conjugates. Under normal culture conditions, approximately 10% of the injected ubiquitin is linked to histones, 40% is found in conjugates with molecular weights greater than 25,000, and the rest is unconjugated. After heat-shock, the free ubiquitin pool and the level of histone-ubiquitin conjugates decreased rapidly, and high molecular weight conjugates predominated. Formation of large conjugates did not require protein synthesis; when analyzed by two-dimensional electrophoresis, the major conjugates did not co-migrate with heat- shock proteins before or after thermal stress. Concomitant with the loss of free ubiquitin, the degradation of endogenous proteins, injected hemoglobin, BSA, and ubiquitin was reduced in heat-shocked HeLa cells. After reversible heat-shock, the decrease in proteolysis was small, and both the rate of proteolysis and the size of the free ubiquitin pool returned to control levels upon incubation at 37 degrees C. In contrast, neither proteolysis nor free ubiquitin pools returned to control levels after lethal heat-shock. However, lethally heat- shocked cells degraded denatured hemoglobin more rapidly than native hemoglobin and ubiquitin-globin conjugates formed within them. Therefore, stabilization of proteins after heat-shock cannot be due to the loss of ubiquitin conjugation or inability to degrade proteins that form conjugates with ubiquitin.
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DOI：
10.1073/pnas.81.21.6779
发表时间：
1984-01-01
期刊：
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA-BIOLOGICAL SCIENCES
影响因子：
--
作者：
BAKER, TA;GROSSMAN, AD;GROSS, CA
通讯作者：
GROSS, CA
影响因子：
4.1
作者：
DAHLMANN, B;KUEHN, L;REINAUER, H
通讯作者：
REINAUER, H
DOI：
10.1073/pnas.83.1.90
发表时间：
1986-01-01
影响因子：
11.1
作者：
LEICHT, BG;BIESSMANN, H;BONNER, JJ
通讯作者：
BONNER, JJ
影响因子：
56.9
作者：
ANANTHAN, J;GOLDBERG, AL;VOELLMY, R
通讯作者：
VOELLMY, R
DOI：
10.1073/pnas.81.21.6647
发表时间：
1984-01-01
期刊：
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA-BIOLOGICAL SCIENCES
影响因子：
--
作者：
GOFF, SA;CASSON, LP;GOLDBERG, AL
通讯作者：
GOLDBERG, AL