The aim of this study is to define and to characterize those factors which influence the assembly of chlorophyll a/b binding polypeptides (CAs polypeptides; into the light harvesting complexes associated with photosystem I (LHC I) and photosystem II (LHCII). Assembly of CAB polypeptides into LHC involves a series of complicated processes that include apoprotein insertion into the membrane, attachment to pigments and assembly into either LHC I or LHC II asymmetrically located in the membrane. In addition PS II- CAs polypeptides redistribute themselves within the membrane in response to environmental conditions.
Our aim i s to dissect these complicated processes into understandable and predictable biochemical reactions. We will develop a system in isolated chloroplasts for defining protein domains involved in the assembly of a given CAB polypeptide into LHC I versus LHC II. Chimaeric genes will be constructed between PS I and PS II cab genes. These constructs will be transcribed and translated in vitro and the resulting radiolabeled fusion proteins will be imported, processed and assembled into LHC in organello. Subsequent fractionation of the chloroplasts into photosystem. I and II will enable us to determine which protein domains mediate photosystem specific assembly. We will further exploit the in organello system to test whether phosphorylation of the precursor affects the distribution of the polypeptides into appressed or nonappressed membranes. This question is of relevance because phosphorylation-mediated redistribution of CAB polypeptides is thought to influence the relative excitation of is I and PS II. To complement our in organello approach, we will attempt to develop a subchloroplast system in which we can reconstitute the assembly pathway. Presently a system exists in which a single CAB polypeptide has been inserted in isolated thylakoid membranes supplemented with a stromal extract. We will extend such studies to include the insertion of a wide range of CAB polypeptides, the involvement of fatty acid acylation in this process, and the requirements of pigments for assembly. We will attempt to reconstitute assembly in subthylakoid systems using membranes enriched in either PS I or PS II and artificial membranes reconstituted with reaction centers devoid of LHC. Such studies will help to characterize the machinery required for the integration of CAB polypeptides into the membrane and their assembly into LHC. The aforementioned approaches should help us understand how to account for the nonrandom distribution of proteins within physically continuous membranes.
