EVIDENCE FOR NUCLEAR TARGETING OF PROTHYMOSIN AND PARATHYMOSIN SYNTHESIZED INSITU
EVIDENCE FOR NUCLEAR TARGETING OF PROTHYMOSIN AND PARATHYMOSIN SYNTHESIZED INSITU
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DOI：
10.1073/pnas.88.15.6608
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发表时间：
1991-08-01
影响因子：
11.1
通讯作者：
HORECKER, BL
中科院分区：
文献类型：
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作者：
CLINTON, M;GRAEVE, L;HORECKER, BL
为了验证原胸腺酶和副胸腺酶含有导致其靶向细胞核的氨基酸序列的假设，构建了包含类人猿病毒40启动子和编码由截断的人生长激素（hGH）组成的嵌合蛋白与原胸腺酶或副胸腺酶的NH2末端连接的cdna的表达载体。截断的hGH缺乏分泌所需的信号肽序列。将这些构建体转染到正常情况下不合成hGH的HeLa S3细胞后，使用间接免疫荧光染色跟踪hGH嵌合体的定位表明，前胸腺酶和副胸腺酶都引起了对细胞核的靶向。一个编码天然生长激素的结构体和一个编码缺乏信号肽的截断生长激素的结构体的对照，在第一种情况下显示分泌到培养基中并在内质网和高尔基体中染色，在第二种情况下弥漫染色整个细胞质。通过原位合成的蛋白质，研究结果为胸腺酶原和副胸腺酶中存在核定位信号提供了直接证据。
To test the hypothesis that prothymosin and parathymosin contain amino acid sequences that cause them to be targeted to the cell nucleus, expression vectors were constructed containing a simian virus 40 promoter and cDNAs that would code for chimeric proteins composed of truncated human growth hormone (hGH) linked to the NH2 terminus of prothymosin or parathymosin. The truncated hGH lacked the signal peptide sequence required for its secretion. After transfection of these constructs into HeLa S3 cells, which do not normally synthesize hGH, the use of indirect immunofluorescence staining to follow the localization of the hGH chimeras demonstrated that both prothymosin and parathymosin caused targeting to the cell nucleus. Controls with a construct coding for native hGH only, and one coding for the truncated hGH lacking the signal peptide, revealed secretion into culture medium and staining in the endoplasmic reticulum and Golgi apparatus in the first case, and diffuse staining throughout the cytoplasm in the second. The results provide direct evidence, with proteins synthesized in situ, for the presence of nuclear localization signals in both prothymosin and parathymosin.