Abstract
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.
Highlights
The insertion of membrane proteins is a complex process in which two major obstacles must be overcome: the transfer of hydrophobic regions into the bilayer with the correct final topology and the efficient translocation of hydrophilic domains to the trans side of the bilayer
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 light-harvesting chlorophyll-binding protein (LHCP)
We show that photosystem II subunit S (PsbS) and early light-inducible protein 2 (Elip2) molecules are capable of following either “unassisted” or assisted pathways, and we discuss the implications for the mechanism and role of signal recognition particle (SRP) in chloroplasts
Summary
The insertion of membrane proteins is a complex process in which two major obstacles must be overcome: the transfer of hydrophobic regions into the bilayer with the correct final topology and the efficient translocation of hydrophilic domains to the trans side of the bilayer Many such proteins are believed to insert post-translationally into bacterial, mitochondrial, and chloroplast membranes, and a variety of in vivo and in vitro approaches have been used to study the underlying mechanisms. One imported multispanning membrane protein, the major light-harvesting chlorophyll-binding protein of photosystem II (Lhcb, but usually termed LHCII or LHCP) has been shown to require SRP together with GTP for insertion into the membrane [17], and proteolysis of thylakoids blocks this insertion process [18, 19] indicating the involvement of proteintargeting apparatus (probably the Sec apparatus, but this remains to be confirmed). Sec/SRP-independent Protein Insertion into Thylakoids may exist because no RNA molecule has been identified in stromal SRP, and the SRP54 subunit instead forms a complex with a novel 43-kDa subunit [20]
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