BIOGENESIS OF A PHOTOSYSTEM-1 LIGHT-HARVESTING COMPLEX - EVIDENCE FORA MEMBRANE INTERMEDIATE

CAB-7p is a chlorophyll a/b binding protein of photosystem I (PSI). It is found in light-harvesting complex I 680 (LHCI-680), one of the chl orophyll complexes produced by detergent solubilization of PSI. Two ty pes of evidence are presented to indicate that assembly of CAB-7p into PSI proceeds through a membrane intermediate. First, when CAB-7p is b riefly imported into chloroplasts or isolated thylakoids, we initially observe a fast-migrating membrane form of CAB-7p that is subsequently converted into PSI. The conversion of the fast-migrating form into PS I does not require stroma or ATP. Second, trypsin treatment of thylako ids containing radiolabeled CAB-7p indicates that there are at least t wo membrane forms of the mature 23-kD protein. The predominant form is completely resistant to proteolysis; a second form of the protein is cleaved by trypsin into 12- and 7-kD polypeptides. We interpret this t o mean that the intermediate is a cleavable form that becomes protease resistant during assembly. This notion is supported by the observatio n that CAB-7p in LHCI-680 is largely cleaved by trypsin into 12- and 7 -kD polypeptides, whereas CAB-7p in isolated PSI particles is trypsin resistant. In vitro, we generated a mutant form of CAB-7p, CAB-7/Bgl2p , that was able to integrate into thylakoid membranes but was unable t o assemble into PSI. The membrane form of CAB-7/Bgl2p, like LHCI-680, was predominantly cleaved by trypsin into 12- and 7-kD fragments. We s uggest that the mutant protein is arrested at an intermediate stage in the assembly pathway of PSI. Based on its mobility in nondenaturing g els and its susceptibility to protease cleavage, we suggest that the i ntermediate form is LHCI-680. We propose the following distinct stages in the biogenesis of LHCI: (a) apoprotein is integrated into the thyl akoid, (b) chlorophyll is rapidly bound to apoprotein forming LHCI-680 , and (c) LHCI-680 assembles into the native PSI complex.