Optimized APEX2 peroxidase-mediated proximity labeling in fast- and slow-growing mycobacteria

Abstract

Proximity labeling is a technology for tagging proteins and other biomolecules in living cells. These methods use enzymes that generate reactive species whose properties afford high spatial resolution for the localization of proteins to subcellular compartments and the identification of endogenous interaction partners. Here we present the adaptation of the engineered peroxidase APEX2 to proximity labeling in mycobacteria, including the human pathogen Mycobacterium tuberculosis. APEX2 is uniquely suited for general use in bacteria because unlike other proximity labeling enzymes, it does not depend on metabolites like ATP that are found in the cytoplasm, but are absent from the bacterial periplasm. Importantly, we found that in slow-growing mycobacteria like M. tuberculosis, codon usage optimization is required for APEX2 export into the periplasm via fusion to an N-terminal secretion signal. APEX2 expressed from codon-optimized genes affords robust, compartment-specific protein labeling in the cytoplasm and the periplasm of both fast- and slow-growing species. Here we detail these updated constructs and provide an optimized protocol for APEX2-mediated protein labeling in mycobacteria. We expect this approach to be broadly useful for determining the localization of specific proteins, cataloging subcellular proteomes, and identifying interaction partners of 'bait' proteins expressed as fusions to APEX2.

Keywords: APEX2; Mycobacteria; Periplasm; Peroxidase; Protein labeling; Proximity labeling; Tuberculosis.