Elucidating the function of the Bcl2-associated athanogene 6 (BAG6) protein in Chlamydomonas

• Applicant: Professor Dr. Michael Schroda
• Subject Area: Plant Biochemistry and Biophysics ; Plant Physiology
• Term: since 2024
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 536728273

The BAG (B cell lymphoma 2 (Bcl-2)-associated athanogene) family is an evolutionarily conserved group of proteins that share a common conserved region known as the BAG domain. BAG proteins play important roles as molecular co-chaperones of HSP70 in various signal transduction pathways. Via their BAG domain, they bind to the ATPase domain of HSP70 and modulate its chaperone activity. Some plant BAGs contain additional domains/motifs unique to plants such as the calmodulin-binding IQ motif located near the BAG domain. However, the roles of BAG proteins in plant protein homeostasis and associated Ca2+ signaling are poorly understood. While Arabidopsis encodes seven BAG family members (BAG1-7), Chlamydomonas reinhardtii encodes only a single BAG protein, which is homologous to Arabidopsis BAG6, the largest member of the plant BAG family. Chlamydomonas BAG6 caught our attention when we found it in a large-scale proteomics study to be strongly upregulated during a 3-h heat stress time course. qRT-PCR analyses revealed BAG6 transcripts to accumulate upon heat stress even more rapidly than transcripts of small heat shock protein genes HSP22E/F, which serve as sensitive stress markers in Chlamydomonas. We found BAG6 gene expression to be regulated by heat shock factor 1, which rapidly bound to the BAG6 promoter and triggered histone H3/4 acetylation and histone H3 release. An antibody raised against BAG6 revealed that BAG6 protein was not detectable under ambient conditions but accumulated as rapidly as BAG6 transcripts upon heat stress. BAG6 protein levels declined with similar kinetics as sHSP’s and CLPB’s during recovery from heat stress, indicating active degradation of BAG6 and tight control of BAG6 levels. We obtained a putative bag6 knock-out mutant from a Chlamydomonas insertion mutant library and could show that heat-induced BAG6 protein accumulation is abolished in this mutant. Surprisingly, we observed an increased accumulation of HSP22E/F and of protein aggregates in the bag6 mutant when compared with the wild type, suggesting that BAG6 acts as repressor of sHSP accumulation. Co-immunoprecipitation analyses revealed cytosolic HSP70A and HSP22A as BAG6 interaction partners. The goal of the proposed project is to shed more light on the role of BAG6 in regulating protein homeostasis in Chlamydomonas. To this end, we want to identify the stress conditions triggering BAG6 protein accumulation in order to find conditions leading to growth phenotypes in the bag6 mutant, lines harboring BAG6 without IQ motif, and lines constitutively overaccumulating BAG6. We want to get a comprehensive picture on genes/protein dysregulated in the absence/constitutive presence of BAG6. We want to localize BAG6 in the cell and want to identify transient and stable interactions partners in addition to HSP70A/HSP22A. Finally, we want to reconstitute the HSP70A-BAG6-HSP22A chaperone system in vitro, including the newly identified partners.

• DFG Programme: Research Grants