EVIDENCE FOR A STROMAL GTP REQUIREMENT FOR THE INTEGRATION OF A CHLOROPHYLL A B-BINDING POLYPEPTIDE INTO THYLAKOID MEMBRANES/

The integration of chlorophyll a/b-binding (LHCP) polypeptides and the translocation of the 33-kD oxygen-evolving enhancer protein (OEE33) h ave 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 transla tion products depleted of low molecular weight compounds. LHCP integra tion 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 LHC P integration activity than GTP alone, suggesting that GTP meets the e ntire nucleotide requirement. Nonhydrolyzable analogs or GTP were inhi bitory, consistent with the idea that GTP hydrolysis is required for i ntegration activity. Periodate-oxidized GTP (GTPox) also inhibited the integration reaction when present during the assay. Pretreatment of s troma with GTPox followed by GTPox removal inhibited integration activ ity, whereas pretreatment of thylakoids had no effect. We interpret th is to mean that a GTP-binding protein involved in integration is local ized in the stroma. Translocation of OEE33 was more efficient with ATP than with GTP, and the combination of both nucleotides was not additi ve. Our data implicate the involvement of a GTPase in LHCP integration but not in the translocation of OEE33.