Mycobacterial resistance to zinc poisoning requires assembly of P-ATPase-containing membrane metal efflux platforms

Abstract

The human pathogen Mycobacterium tuberculosis requires a P1B-ATPase metal exporter, CtpC (Rv3270), for resistance to zinc poisoning. Here, we show that zinc resistance also depends on a chaperone-like protein, PacL1 (Rv3269). PacL1 contains a transmembrane domain, a cytoplasmic region with glutamine/alanine repeats and a C-terminal metal-binding motif (MBM). PacL1 binds Zn2+, but the MBM is required only at high zinc concentrations. PacL1 co-localizes with CtpC in dynamic foci in the mycobacterial plasma membrane, and the two proteins form high molecular weight complexes. Foci formation does not require flotillin nor the PacL1 MBM. However, deletion of the PacL1 Glu/Ala repeats leads to loss of CtpC and sensitivity to zinc. Genes pacL1 and ctpC appear to be in the same operon, and homologous gene pairs are found in the genomes of other bacteria. Furthermore, PacL1 colocalizes and functions redundantly with other PacL orthologs in M. tuberculosis. Overall, our results indicate that PacL proteins may act as scaffolds that assemble P-ATPase-containing metal efflux platforms mediating bacterial resistance to metal poisoning.

Modulation of bacterial membrane proteins activity by clustering into plasma membrane nanodomains

“…PacL1 is composed of a transmembrane (TM) N-terminal segment, a cytoplasmic region harboring Glu/ Ala (EA) repeats and exhibiting the conserved DUF1490 domain, and a C-terminal intrinsically disordered region (IDR) with a zinc metal-binding motif. We showed that PacL1 is essential for CtpCdependent zinc tolerance in vivo (Boudehen et al, 2022) Altogether, these observations suggest the existence of clusters of metal efflux pumps, which we named metal efflux platforms, organized in the membrane by the PacL proteins (Boudehen et al, 2022).…”

“…Some may rely on physical properties such as phase separation (Heinkel et al, 2019), membrane curvature (Saaki et al, 2018), or preferential interactions with specific lipids, such as those forming lipid domains (Alvarez & Georgellis, 2019; Barák & Muchová, 2013) or the carotenoids enriched in FMM (García‐Fernández et al, 2017; López & Kolter, 2010). Some nanodomains can form through protein–protein interactions with specific scaffold proteins such as flotillins or the PacL proteins (Boudehen et al, 2022; García‐Fernández et al, 2017). The emergence of this new field will greatly enhance our understanding of membrane biology and its integration in bacterial physiology.…”

“…Of note, PacL1 is able to bind zinc through its C-terminal metalbinding motif. However, although ablation of this motif totally abolished the binding of zinc, it only impaired resistance to elevated concentrations of metal, but not to intermediate concentrations (Boudehen et al, 2022).…”

“…PacL1 is composed of a transmembrane (TM) N‐terminal segment, a cytoplasmic region harboring Glu/Ala (EA) repeats and exhibiting the conserved DUF1490 domain, and a C‐terminal intrinsically disordered region (IDR) with a zinc metal‐binding motif. We showed that PacL1 is essential for CtpC‐dependent zinc tolerance in vivo (Boudehen et al, 2022). PacL1 directly interacts with CtpC, and both proteins are found in high‐molecular‐weight complexes in the membrane.…”

“…Western blot analysis of tagged PacL1 and CtpC demonstrated that PacL1 is necessary for the stability of CtpC in the membrane (Figure 1c). Modifications of the EA motifs of PacL1 abolished the direct interaction of PacL1 to the N-terminal domain of CtpC and resulted in destabilization of CtpC(Boudehen et al, 2022). Therefore, PacL1 appears as a chaperon-like protein necessary for CtpC clustering and stability in the membrane.Two other P-type ATPases of M. tuberculosis, namely CtpG(López et al, 2018) and CtpV(Ward et al, 2010), are also encoded in operons with small proteins, very similar to PacL1, namely Rv1993cor PacL2 and Rv0968 or PacL3, respectively.…”