Interplay of four auxiliary factors is required for the assembly of photosystem I reaction center subcomplex.

The photosystem I (PSI) complex consisting of reaction center (RC) subunits, several peripheral subunits, and many cofactors, is present in the thylakoid membranes of chloroplasts and cyanobacteria. The assembly of RC subunits (PsaA/B) which bind electron transfer cofactors and antenna pigments is an intricate process and is mediated by several auxiliary factors such as Ycf3, Y3IP1/CGL59, Ycf4, and Ycf37/PYG7/CGL71. However, their precise molecular mechanisms in RC assembly remain to be addressed. Here we purified four PSI auxiliary factors by affinity chromatography and characterized copurified PSI assembly intermediates. We suggest that Ycf3 assists the initial assembly of newly synthesized PsaA/B subunits into an RC subcomplex while Y3IP1 may be involved in transferring the RC subcomplex from Ycf3 to the Ycf4 module which stabilizes it. CGL71 may form an oligomer that transiently interacts with the PSI RC subcomplex, physically protecting it under oxic conditions until association with the peripheral PSI subunits occurs. Together, our results reveal the interplay among four auxiliary factors required for the stepwise assembly of the PSI RC.