The Accessory SecA2 System of Mycobacteria Requires ATP Binding and the Canonical SecA1

Abstract

In bacteria, the majority of exported proteins are transported by the general Sec pathway from their site of synthesis in the cytoplasm across the cytoplasmic membrane. The essential SecA ATPase powers this Sec-mediated export. Mycobacteria possess two nonredundant SecA homologs: SecA1 and SecA2. In pathogenic Mycobacterium tuberculosis and the nonpathogenic model mycobacterium Mycobacterium smegmatis, SecA1 is essential for protein export and is the "housekeeping" SecA, whereas SecA2 is an accessory SecA that exports a specific subset of proteins. In M. tuberculosis the accessory SecA2 pathway plays a role in virulence. In this study, we uncovered basic properties of the mycobacterial SecA2 protein and its pathway for exporting select proteins. By constructing secA2 mutant alleles that encode proteins defective in ATP binding, we showed that ATP binding is required for SecA2 function. SecA2 mutant proteins unable to bind ATP were nonfunctional and dominant negative. By evaluating the subcellular distribution of each SecA, SecA1 was shown to be equally divided between cytosolic and cell envelope fractions, whereas SecA2 was predominantly localized to the cytosol. Finally, we showed that the canonical SecA1 has a role in the process of SecA2-dependent export. The accessory SecA2 export system is important to the physiology and virulence of mycobacteria. These studies help establish the mechanism of this new type of specialized protein export pathway.

Clostridium difficile Has Two Parallel and Essential Sec Secretion Systems

“…6); SecA2 was found predominantly in the soluble fraction with only a small amount of protein detected at the membrane, whereas SecA1 was detected only at the membrane. This is in stark contrast to M. smegmatis, where SecA1 was found to be localized equally between the membrane and cytoplasm (31). However, as was observed in M. smegmatis, the dominant-negative K106R mutation in SecA2 switched the localization of the protein entirely to the membrane fraction.…”

“…Cellular Distribution of SecA1 and SecA2-It has been previously reported that M. smegmatis SecA1 and SecA2 differ in their subcellular localization; SecA1 was found equally in cytoplasmic and membrane fractions, whereas SecA2 was predominantly cytoplasmic (31). Furthermore, the SecA2(K129R) mutation, analogous to the SecA2(K106R) mutation described in this study, relocalized SecA2 predominantly to the membrane.…”

“…However, we took advantage of a previous observation that a conservative Lys-to-Arg substitution in the Walker box of SecA, which negates binding to ATP, confers a dominant-negative phenotype in both E. coli and M. smegmatis (31,44). In M. smegmatis, overexpression of dominant-negative SecA2(K129R) gave the same phenotype as a secA2 knock-out (31). Lys-129 corresponds to Lys-106 in both SecA1 and SecA2 of C. difficile (supplemental Fig.…”

“…In M. smegmatis, SecA1 and SecA2 have non-redundant functions in protein translocation; SecA1 provides essential housekeeping functions, whereas SecA2 is responsible for the secretion of a small number of lipoproteins (30). Furthermore, the M. smegmatis accessory pathway also appears to require SecA1 (31).…”

“…Of the organisms possessing a SecA2-only accessory Sec system, Mycobacterium smegmatis is perhaps the most intensively studied (26,30,31). In M. smegmatis, SecA1 and SecA2 have non-redundant functions in protein translocation; SecA1 provides essential housekeeping functions, whereas SecA2 is responsible for the secretion of a small number of lipoproteins (30).…”