Identification of two HSP70-related Xenopus oocyte proteins that are capable of recycling across the nuclear envelope.
Identification of two HSP70-related Xenopus oocyte proteins that are capable of recycling across the nuclear envelope.
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DOI：
10.1083/jcb.111.5.1775
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发表时间：
1990-11
期刊：
影响因子：
--
通讯作者：
Feldherr CM
中科院分区：
文献类型：
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作者：
Mandell RB;Feldherr CM
两种70kD的多肽B3和B4在非洲爪哇卵母细胞的胞核和胞浆中以相同的浓度存在。这项研究的目的是确定它们(A)是否是70kD热休克蛋白家族的成员，以及(B)在核间和细胞质间循环。基于与ATP的高亲和力结合、B3/B4特异性抗体与大鼠hsc70的交叉反应以及与hsc70的溴化氰裂解肽图谱的比较，证实了B3和B4是热休克蛋白70-kD家族的成员。通过将125I标记的B3/B4、大鼠hsc70和BSA显微注射到卵母细胞的细胞质中，并检测它们随后在细胞内的分布，进行了核摄取研究。在注射后6h，B3/B4和hsc70的核浓度大约是BSA对照组的24倍。研究还发现，直径高达120A的B3/B4包覆的金粒子能够通过孔洞进入原子核。将碘化蛋白直接注射到卵母细胞核中，分析核外排。注射后2 h，胞浆中至少有46%的B3/B4和60%的hsc70存在于胞浆中，而BSA对照组的B3/B4和hsc70的含量不到10%。在细胞融合实验中，标记的、无核的卵母细胞植物半球在油中与有核的、未标记的动物半球融合，证明细胞质B3和B4在达到平衡后可以进入细胞核，这证明了核群和细胞质群的分离存在。总而言之，这些结果表明，B3、B4和大鼠hsc70跨核膜运输，并在细胞核和细胞质之间循环。
Two 70-kD polypeptides, B3 and B4, are present in equivalent concentrations in the nucleus and cytoplasm of Xenopus oocytes. The objectives of this study were to determine if they (a) are members of the 70-kD family of heat shock proteins, and (b) recycle between the nuclear and cytoplasmic compartments. Evidence based on high-affinity binding to ATP, cross-reactivity of B3/B4-specific antibodies with rat hsc70, and a comparison of cyanogen bromide cleavage peptide maps with hsc70, verified that B3 and B4 are members of the 70-kD family of heat- shock proteins. Nuclear uptake studies were performed by microinjecting 125I-labeled B3/B4, rat hsc70, and BSA into the cytoplasm of oocytes, and examining their subsequent intracellular distributions. By 6 h postinjection, the nuclear concentration of B3/B4 and hsc70 were approximately 24-fold greater than BSA controls. It was also found that B3/B4-coated gold particles as large as 120A in diameter were able to enter the nucleus by passing through the pores. Nuclear efflux was analyzed by microinjecting the iodinated proteins directly into the oocyte nuclei. 2 h after nuclear injection, at least 46% of the B3/B4 and 60% of the hsc70 were found in the cytoplasmic fractions, compared with less than 10% for the BSA controls. Cell fusion experiments, in which labeled, anucleate oocyte vegetal hemispheres were fused, under oil, with nucleate unlabeled animal hemispheres, demonstrated that cytoplasmic B3 and B4 could enter the nucleus after equilibration was reached, arguing against the existence of separate nuclear and cytoplasmic populations. Collectively, these results show that B3, B4, and rat hsc70 are transported across the nuclear envelope and recycle between the nucleus and cytoplasm.