Evidence for a Stromal GTP Requirement for the Integration of a Chlorophyll a/b-Binding Polypeptide into Thylakoid Membranes

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Abstract
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.