Hoffman N E, Franklin A E
Department of Plant Biology, Carnegie Institution of Washington, Stanford, California.
Plant Physiol. 1994 May;105(1):295-304. doi: 10.1104/pp.105.1.295.
The integration of chlorophyll a/b-binding (LHCP) polypeptides and the translocation of the 33-kD oxygen-evolving enhancer protein (OEE33) have been previously shown to occur in chloroplast extracts containing stroma, thylakoids, ATP, and MgCl2. We have re-examined the nucleotide requirement for these two reactions using stromal extract and translation products depleted of low molecular weight compounds. LHCP integration activity was up to 10-fold higher when assayed with GTP compared with ATP, CTP, or UTP. A combination of ATP and GTP supported less LHCP integration activity than GTP alone, suggesting that GTP meets the entire nucleotide requirement. Nonhydrolyzable analogs of GTP were inhibitory, consistent with the idea that GTP hydrolysis is required for integration activity. Periodate-oxidized GTP (GTPox) also inhibited the integration reaction when present during the assay. Pretreatment of stroma with GTPox followed by GTPox removal inhibited integration activity, whereas pretreatment of thylakoids had no effect. We interpret this to mean that a GTP-binding protein involved in integration is localized in the stroma. Translocation of OEE33 was more efficient with ATP than with GTP, and the combination of both nucleotides was not additive. Our data implicate the involvement of a GTPase in LHCP integration but not in the translocation of OEE33.
叶绿素a/b结合（LHCP）多肽的整合以及33-kD放氧增强蛋白（OEE33）的转运先前已表明发生在含有基质、类囊体、ATP和MgCl2的叶绿体提取物中。我们使用去除了低分子量化合物的基质提取物和翻译产物重新研究了这两个反应对核苷酸的需求。与ATP、CTP或UTP相比，用GTP检测时LHCP整合活性高出10倍。ATP和GTP的组合支持的LHCP整合活性比单独使用GTP时要低，这表明GTP满足了整个核苷酸需求。GTP的不可水解类似物具有抑制作用，这与整合活性需要GTP水解的观点一致。在检测过程中存在高碘酸盐氧化的GTP（GTPox）时也会抑制整合反应。用GTPox预处理基质然后去除GTPox会抑制整合活性，而对类囊体进行预处理则没有影响。我们将此解释为参与整合的GTP结合蛋白定位于基质中。OEE33的转运使用ATP比使用GTP更有效，并且两种核苷酸的组合没有加成作用。我们的数据表明GTP酶参与LHCP的整合，但不参与OEE33的转运。
