Expression and processing of proteins encoded by the Saccharomyces retrotransposon Ty5
Expression and processing of proteins encoded by the Saccharomyces retrotransposon Ty5
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DOI：
10.1128/jvi.75.4.1790-1797.2001
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发表时间：
2001-02-01
影响因子：
5.4
通讯作者：
Voytas, DF
中科院分区：
文献类型：
--
作者：
Irwin, PA;Voytas, DF
逆转录元件(逆转录转座子和逆转录病毒)有两个共同的基因：GAG和POL，前者指定形成病毒或病毒样颗粒的结构蛋白，后者指定复制所需的催化蛋白。对于许多反转录元件，Gag和Pol分别出现在不同的读框上。它们的表达受到高度调控，GAG/POL的比例对逆转录元件复制至关重要。酵母反转录转座子TY5含有一个单一的开放阅读框架，我们通过产生N端或C端或整合酶编码区内带有表位标签的转座活性TY5元件来表征Gag和Pol的表达。免疫印迹分析鉴定出两种Gag物种(Gag-P27和Gag-P37)、逆转录酶(Pol-P59)和整合酶(Pol-P80)，它们在没有尿素或离子洗涤剂的情况下都很难溶解。这些蛋白是多聚蛋白的蛋白水解性处理的结果，因为在TY5蛋白酶假定活性部位发生突变的元件表达单一标记蛋白(Gag-Pol-p182)。蛋白水解酶突变体也是转位不活跃的。在时间进程实验中，我们监测了在N端和C端具有相同表位标签的TY5元件的蛋白质表达、蛋白降解处理和转座。转座和Gag-p27的丰度都随着时间的推移而增加。相反，GAG-p37和逆转录酶在诱导后14h达到峰值，然后逐渐下降。这可能是由于GAG-p27与GAG-P37和逆转录酶的稳定性不同所致。在整个时间过程实验中，TY5 Gag与Pol的比例平均为5：1，这表明差异蛋白质的稳定性调节了这些蛋白质的数量。
Retroelements (retrotransposons and retroviruses) have two genes in common: gag, which specifies structural proteins that form a virus or virus-like particle, and pol, which specifies catalytic proteins required for replication. For many retroelements, gag and pol are present on separate reading frames. Their expression is highly regulated, and the ratio of Gag to Pol is critical for retroelement replication. The Saccharomyces retrotransposon Ty5 contains a single open reading frame, and we characterized Gag and Pol expression by generating transpositionally active Ty5 elements with epitope tags at the N terminus or C terminus or within the integrase coding region. Immunoblot analysis identified two Gag species (Gag-p27 and Gag-p37), reverse transcriptase (Pol-p59), and integrase (Pol-p80), all of which are largely insoluble in the absence of urea or ionic detergent. These proteins result from proteolytic processing of a polyprotein, because elements with mutations in the presumed active site of Ty5 protease express a single tagged protein (Gag-Pol-p182). Protease mutants are also transpositionally inactive. In a time course experiment, we monitored protein expression, proteolytic processing, and transposition of a Ty5 element with identical epitope tags at its N and C termini. Both transposition and the abundance of Gag-p27 increased over time. In contrast, the levels of Gag-p37 and reverse transcriptase peaked after similar to 14 h of induction and then gradually decreased. This may be due to differences in stability of Gag-p27 relative to Gag-p37 and reverse transcriptase. The ratio of Ty5 Gag to Pol averaged 5:1 throughout the time course experiment, suggesting that differential protein stability regulates the amounts of these proteins.