Co-translational Assembly of the D1 Protein into Photosystem II

Lixin Zhang,Virpi Paakkarinen,Klaas Jan Van Wijk,Eva-Mari Aro

doi:10.1074/jbc.274.23.16062

Lixin Zhang, Virpi Paakkarinen + Show 2 more

Open Access

https://doi.org/10.1074/jbc.274.23.16062

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Abstract

Assembly of multi-subunit membrane protein complexes is poorly understood. In this study, we present direct evidence that the D1 protein, a multiple membrane spanning protein, assembles co-translationally into the large membrane-bound complex, photosystem II. During pulse-chase studies in intact chloroplasts, incorporation of the D1 protein occurred without transient accumulation of free labeled protein in the thylakoid membrane, and photosystem II subcomplexes contained nascent D1 intermediates of 17, 22, and 25 kDa. These N-terminal D1 intermediates could be co-immunoprecipitated with antiserum directed against the D2 protein, suggesting co-translational assembly of the D1 protein into PS II complexes. Further evidence for a co-translational assembly of the D1 protein into photosystem II was obtained by analyzing ribosome nascent chain complexes liberated from the thylakoid membrane after a short pulse labeling. Radiolabeled D1 intermediates could be immunoprecipitated under nondenaturing conditions with antisera raised against the D1 and D2 protein as well as CP47. However, when the ribosome pellets were solubilized with SDS, the interaction of these intermediates with CP47 was completely lost, but strong interaction of a 25-kDa D1 intermediate with the D2 protein still remained. Taken together, our results indicate that during the repair of photosystem II, the assembly of the newly synthesized D1 protein into photosystem II occurs co-translationally involving direct interaction of the nascent D1 chains with the D2 protein.

Highlights

Photosystem II (PS II)1 is a multiprotein membrane complex that catalyzes water oxidation and reduction of plastoquinone
Insertion of Newly Synthesized D1 Protein into PS II Subcomplexes in Intact Chloroplasts—In earlier studies we observed that the incorporation of the newly synthesized D1 protein into PS II core complexes proceeded in a stepwise manner [17]
We postulated that the assembly of the D1 protein occurs mainly in a post-translational pathway in which the full-length D1 first accumulated as an unassembled protein, in parallel to a possible co-translational pathway in which D1 incorporates directly into PS II reaction centers [16]

Summary

Introduction

Photosystem II (PS II)1 is a multiprotein membrane complex that catalyzes water oxidation and reduction of plastoquinone. Analysis of pulse labeled thylakoids (2.5-min pulse and no chase) by a combination of the optimized sucrose gradient fractionation and nondenaturing Deriphat-PAGE (Fig. 2) revealed a rapid assembly of most newly synthesized proteins into PS II subcomplexes.

Results

Conclusion