The assembly of the PsaD subunit into the membranal photosystem I complex occurs via an exchange mechanism

PsaD is a peripheral stromal-facing subunit of photosystem I (PSI), a multi subunit complex of the thylakoid membranes. PsaD plays a major role in both the function and assembly of PSI. Past studies with radiolabeled PsaD indi cated that PsaD is able to assemble in vitro specifically into the PSI comp lex. To unravel the mechanism by which this assembly takes place, the follo wing steps were taken. (i) Mature PsaD of spinach and PsaD of the prokaryot ic caynobacterium Mastigocladus laminosus, both bearing a six-histidine tag at their C-termini, were overexpressed in Escherichia coli and purified to homogeneity. (ii) The purified recombinant protein was introduced into the isolated PSI complex. (iii) Following incubation, the PsaD that assembled into PSI was separated from the nonassembled PsaD by a sucrose gradient. Di fferential Western blot analysis was used to determine whether the native a nd the recombinant PsaD were present as free or assembled proteins of the P SI complex. Antibodies that can recognize only the recombinant PsaD (anti-h is) or both the native and recombinant PsaD (anti-PsaD) were used. The find ings indicated that an exchange mechanism enables the assembly of a newly i ntroduced PsaD into PSI. The latter replaces the PsaD subunit that is prese nt in situ within the complex. In vivo studies that followed the assembly o f PsaD in Chlamydomonas reinhardtii cells supported this in vitro-character ized exchange mechanism. In C. reinhardtii, in the absence of synthesis and assembly of new PSI complexes, newly synthesized PsaD assembled into pre-e xisting PSI complexes.