Genetically Encodable Bacterial Flavin Transferase for Fluorogenic Protein Modification in Mammalian Cells

Area of Science:

• Synthetic biology
• Molecular biology
• Cellular biology

Background:

• A bacterial flavin transferase, ApbE, mediates flavin mononucleotide (FMN) modification of the Na⁺-translocating NADH:quinone oxidoreductase C (NqrC) protein.
• This unique bacterial post-translational modification has not been previously explored in mammalian systems.

Purpose of the Study:

• To investigate the feasibility of ApbE-mediated FMN transfer onto NqrC in live mammalian cells.
• To explore the potential applications of this system for drug screening and protein interaction studies.

Main Methods:

• Genetic targeting of ApbE and NqrC in the cytosol of mammalian cells.
• Expression of NqrC in the endoplasmic reticulum (ER) to study its cellular fate.
• Utilizing a truncated RnfG (half-RnfG) as a genetically encoded tag for protein interaction monitoring.

Main Results:

• Robust FMN transfer occurred when ApbE and NqrC were co-expressed in the mammalian cell cytosol.
• NqrC expressed in the ER underwent retro-translocation to the cytosol, followed by proteasome-mediated degradation.
• The NqrC-ER system demonstrated improved performance for in cellulo proteasome inhibitor screening.
• Half-RnfG proved effective as a genetically encoded tag for monitoring protein-protein interactions within specific cellular compartments, including the ER.

Conclusions:

• The ApbE-NqrC system is functional in mammalian cells, enabling FMN modification.
• The NqrC-ER-induced degradation pathway can be leveraged for developing novel proteasome inhibitor screening platforms.
• Genetically encoded tags like half-RnfG, modified by ApbE, offer a versatile tool for studying protein interactions in live cells, with implications for anticancer drug screening and synthetic biology.