Biochemical characterization of protein composition and protein phosphorylation patterns in stacked and unstacked thylakoid membranes of the prochlorophyte Prochlorothrix hollandica.

We describe a procedure to separate stacked and unstacked membranes from the prochlorophyte Prochlorothrix hollandica that is based on methods used for the separation of grana and stroma thylakoids from chloroplasts. Stacked membranes were isolated from Triton X-100-treated whole thylakoid preparations, unstacked membranes from French press disrupted cells. Membrane fractions were isolated by differential centrifugation. The stacked membranes were enriched in photosystem (PS) II and a chlorophyll a/b-binding antenna complex, whereas the unstacked membranes contained PS I and the ATP synthase. No evidence for a PS I-associated chlorophyll a/b antenna system was obtained. The PS II-associated chlorophyll a/b antenna complex is composed of several apoproteins in the molecular mass range from 32 to 38 kDa. The 38-kDa protein of this complex becomes phosphorylated on its stromal surface by a light-activated kinase, but, unlike the light-harvesting II complex of chloroplasts, it does not migrate from grana to stroma membrane regions. Overall, the thylakoid membranes of Prochlorothrix exhibit a remarkably similar organization to those of chloroplasts, especially in terms of the non-random distribution of protein complexes between grana and stroma thylakoid membranes. However, in contrast to the rapidly reversible phosphorylation-dependent state 1-state 2 response of light-harvesting complex associated with PS II in chloroplasts, the slowly reversible phosphorylation in Prochlorothrix leads only to a functional uncoupling from PS II but not to its redistribution between stacked and unstacked membrane regions.