Transbilayer organization of the chlorophyll-proteins of spinach thylakoids

To investigate the transverse bilayer organization of the chlorophyll-proteins of the three intrinsic chlorophyll-protein complexes, the effects of proteolytic enzymes, and an antibody against the light-harvesting complex were compared using right-side-out and inside-out thylakoid vesicles. The vesicles were isolated by aqueous polymer phase partitioning following the fragmentation of spinach thylakoids by passage through a Yeda press. Both vesicle types were agglutinated by an antiserum specific for the light-harvesting complex, although proteolytic degradation of the complex occurred only in right-side-out vesicles. In addition, there are different antigenic sites for the light-harvesting complex on the inner and outer thylakoid surfaces. Polypeptides of the chlorophyll-alpha-protein complex of photosystem II were degraded by proteases at both membrane surfaces. We concluded that both these chlorophyll-protein complexes are membrane spanning and transversely asymmetric, but that the light-harvesting complex polypeptides accessible at the inner thylakoid surface are more resistant to proteolytic attack. In contrast, the main chlorophyll-containing polypeptide (Mr = 64 500) of photosystem I complex was resistant to proteolytic attack at both the outer and inner thylakoid surfaces.