Distinct 'assisted' and 'spontaneous' mechanisms for the insertion of polytopic chlorophyll-binding proteins into the thylakoid membrane

The biogenesis of several bacterial polytopic membrane proteins has been shown to require signal recognition particle (SRP) and protein transport machinery, and one such protein, the major light-harvesting chlorophyll- binding protein (LHCP) exhibits these requirements in chloroplasts. In this report we have used in vitro insertion assays to analyze four additional members of the chlorophyll-a/b-binding protein family. We show that two members, Lhca1 and Lhcb5, display an absolute requirement for stroma, nucleoside triphosphates, and protein transport apparatus, indicating an 'assisted' pathway that probably resembles that of LHCP. Two other members, however, namely an early light-inducible protein 2 (Elip2) and photosystem II subunit S (PsbS), can insert efficiently in the complete absence of SRP, SecA activity, nucleoside triphosphates, or a functional Sec system. The data suggest a possibly spontaneous insertion mechanism that, to date, has been characterized only for simple single-span proteins. Of the membrane proteins whose insertion into thylakoids has been analyzed, five have now been shown to insert by a SRP/Sec-independent mechanism, suggesting that this is a mainstream form of targeting pathway. We also show that PsbS and Elip2 molecules are capable of following either 'unassisted' or assisted pathways, and we discuss the implications for the mechanism and role of SRP in chloroplasts.