Genetic and physical interactions with STIC components and SRP54
The negative synergism derived from combining the get3b allele with srp54 , alb4 and stic2 implies an orchestrated network between GET3B and these pathway components probably by ensuring the proper integration and assembly of chloroplast proteins.
In particular, the srp54 get3b double mutant displayed a more severe phenotype than either single mutant by itself (Figure 4), signifying that the srp54 mutation acts as a potentiator of theget3b effect or vice versa. This suggests that SRP54 and GET3B either may work together in a single pathway or may work separately in parallel pathways that converge in similar chloroplastidic processes. Interestingly, SRP54 binds solely to the third TMD of LHCPs (High et al., 1997). This TMD is the most hydrophobic of all three and fusing it alone to a soluble protein will translocate the fusion protein into the thylakoid membrane (Kohorn and Tobin, 1989). Since both SRP54 and GET3B are capable of binding single TMDs, it might be the case that these two proteins are able to partially compensate for the absence of the other, thereby maintaining stromal IMP trafficking when other targeting networks are not fully functional.
The incapacity of the ATPase-deficient variant to rescue the growth phenotype in the alb4 srp54 get3b triple mutant (Figure 5) underscores that it is the targeting function of GET3B rather than the potential holdase function which enables its participation in chloroplastidic proteostasis. The observed growth defects of plants expressing the ATPase-defiant variant in alb4 and stic2 backgrounds (Figure 6) further confirm that operative ATPase activity is essential for a fully functioning GET3B in planta.
The physical interaction between GET3B and the C-terminal regions of ALB3 and ALB4, as demonstrated by pulldown experiments and yeast two-hybrid assays, provides a mechanistic insight into how GET3B may facilitate the insertion of proteins into the thylakoid membrane. Interestingly, irrespective of in vitro or in vivo, both the stromal exposed C-terminal regions or full length ALB3 and ALB4 interacted with GET3B to a fairly similar degree (Figure 7), indicating less selectivity for the insertases than the cpSRP pathway. The C-terminal regions of ALB3 and ALB4 share two conserved motifs: motif I and motif III, which are not utilized by ALB3 for the interaction with SRP43 (Falk et al., 2010). In turn, SRP43 interacts with motifs II and IV of ALB3 which helps it discriminate between the two insertases (Falk et al., 2010; Dünschede et al., 2011). Recently, a direct physical interaction between STIC2 and motif III of ALB3 and ALB4 was demonstrated (Stolle et al., 2024). The plastidic ribosome on the other hand cooperatively binds motif III and IV of ALB3 (Ackermann et al., 2021). In future studies, it would be interesting to characterize the GET3B binding site within the C-terminal regions of the Alb proteins and to investigate whether GET3B and STIC2 may engage in competitive or cooperative binding.
In summary, the results of this study highlight the role of GET3B in chloroplast biogenesis and function. GET3B appears to be involved in the proper assembly and function of photosynthetic complexes but also for proteostasis in a broader sense within the chloroplast. Its genetic interactions with STIC components and SRP54, as well as its dependence on ATPase activity, suggests a critical role in the integration of membrane proteins. Further studies are necessary to explore the precise mechanistic details of GET3B s interactions with the other targeting components and how they contribute to the overall regulation of chloroplast biogenesis and function.