Another key focus of our institute is the assembly of photosystem II (PSII), a central component of the photosynthetic machinery. PSII is responsible for capturing light energy and initiating the conversion of light into chemical energy. Our team has identified a number of eukaryote-specific factors that are crucial for efficient PSII assembly in plants, the molecular function of which we seek to uncover.

We mainly focus on assembly factors involved in the formation of the RC47 intermediate, which results from the association of the reaction center (RC) with the intrinsic antenna protein CP47. Two key proteins, DEAP2 and PAM68, play a critical role in this process. Arabidopsis mutants lacking either of these proteins show reduced growth, pigmentation, and PSII efficiency (Fig. 1, Keller et al., 2023; https://doi.org/10.1093/plphys/kiad446). The deap2 x pam68 double mutants are unable to assemble PSII and therefore cannot fix carbon and only grow when supplemented with sucrose.

Through in vivo labeling experiments, DEAP2 and PAM68 were shown to act in the formation of the RC47 intermediate. In this approach, leaf discs from young leaves of WT, deap2-1 and pam68-2 plants were infiltrated with ³⁵S-labeled methionine. To distinguish newly synthesized proteins, thylakoids were isolated from half of the leaf discs immediately after labeling (pulse), while the remaining discs were washed and infiltrated with non-labeled methionine before thylakoid extraction (chase). The labeled proteins were then visualized using 2D-BN-PAGE (Fig. 2, Keller et al., 2023; https://doi.org/10.1093/plphys/kiad446). These analyses revealed an accumulation of labeled proteins at the RC level in the mutants compared to WT, indicating that the following assembly with CP47 is specifically impaired by the absence of DEAP2 and PAM68.

The exact molecular roles of PAM68 and DEAP2 in the formation of RC47 remain unknown. Interestingly, the function of PAM68 is conserved in photosynthetic autotrophs (Bučinská et al., 2018; doi: 10.1104/pp.18.00061), while DEAP2 is a eukaryote-specific protein. Thus, our group seeks to functionally characterize DEAP2, PAM68 together with other previously undescribed assembly factors involved in the formation of RC47. To this end, we perform in vivo proximity labeling and crosslinking in combination with co-immunoprecipitation. Additionally, we carry out higher order mutant analyses, cross- and subdomain complementation studies.