A conserved membrane protein negatively regulates Mce1 complexes in mycobacteria

Abstract

Tuberculosis (TB) remains one of the deadliest infectious diseases, posing a serious threat to global health. Mycobacterium tuberculosis, the causative agent of TB, is an intracellular pathogen that relies on various mechanisms to survive and persist within the host environment. Among the many virulence factors that contribute to pathogenesis, mycobacteria encode Mce systems, putative transporters that are important for lipid uptake. The molecular basis for Mce function(s) is poorly understood. To gain insights into the composition and architecture of mycobacterial Mce systems, we characterized the putative Mce1 complex involved in fatty acid transport. Using affinity purification, we show that the Mce1 system in Mycobacterium smegmatis is an ATP-binding cassette transporter complex comprising YrbE1A/B as heterodimeric transmembrane domains (TMDs) and MceG as nucleotide-binding domains (NBDs), associated with possible heterohexameric assemblies of MCE-domain substrate-binding proteins. We demonstrate that Msmeg_6540 and Mce1A are functionally redundant homologs, likely forming distinct Mce1 complexes that contribute to fatty acid uptake. Finally, we establish that the membrane protein Msmeg_0959, herein renamed to Mce1N, negatively regulates Mce1 function by blocking MceG access to YrbE1B. This regulatory mechanism is conserved in M. tuberculosis Mce1N (Rv0513). Our work offers molecular understanding of Mce complexes, shedding light on lipid metabolism and its regulation in mycobacteria.

Structure of an endogenous mycobacterial MCE lipid transporter

“…We did not observe any peptides corresponding to mce1 -encoded proteins Mce1R, FadD5, or Mam1A-Mam1D, or the accessory factor LucA. MSMEG_6540, which is 84% identical to Mce1A, but encoded elsewhere in the genome, was also highly enriched in the MceG pull-down and has recently been proposed to play a role in Mce1-mediated fatty acid uptake 34 . While most other mycobacterial MCE proteins are encoded in 6-gene modules, MSMEG_6540 is an “ o rphaned” paralog of Mce1A found in a single gene operon, which we therefore name oMce1A.…”

“…To isolate intact complexes for structural studies in the absence of an established recombinant expression system, we purified endogenous MCE transporters from Msmeg, which have high sequence identity to their Mtb orthologs (~68 % identical 31 ) and similar functions 8,[34][35][36] . We inserted a GFP tag at the C-terminus of MceG in the chromosome of M. smegmatis mc 2 155 using homologous recombination via ORBIT (Fig.…”

“…Quantification of relative protein abundance based on peptide spectral matches shows that Mce1 subunits are most abundant (table S2) ( 30 ). MSMEG_6540, which is 84% identical to Mce1A, was also highly enriched in the MceG pull-down and has recently been proposed to play a role in Mce1-mediated fatty acid uptake ( 26 ). Whereas most other mycobacterial MCE proteins are encoded in 6-gene clusters, MSMEG_6540 is an “orphaned” paralog of Mce1A found in a single gene operon, which we therefore name oMce1A.…”

“…S1B) (23,24), and the proteins from each gene cluster are thought to interact with each other to form large complexes (21). Recombinant expression and purification of MCE complexes has been challenging due to the complexity of their genetic organization, and studies thus far have been limited to single subunits and smaller subcomplexes (25,26). Thus, how proteins are arranged in a complex to facilitate lipid transport across the cell envelope remains unclear, and elucidating the architecture of mycobacterial MCE systems is a key step towards understanding their transport mechanism.…”

Structure of an endogenous mycobacterial MCE lipid transporter

“…1b) 31,32 , and the proteins from each gene cluster are thought to interact with each other to form large complexes 14 . Recombinant expression and puri cation of MCE complexes has been challenging due to the complexity of their genetic organization, and studies thus far have been limited to single subunits and smaller subcomplexes 33,34 . Thus, how proteins are arranged in a complex to facilitate lipid transport across the cell envelope remains unclear, and elucidating the architecture of mycobacterial MCE systems is a key step towards understanding their transport mechanism.…”