Phase separation and clustering of an ABC transporter in Mycobacterium tuberculosis

Abstract

Phase separation drives numerous cellular processes, ranging from the formation of membrane-less organelles to the cooperative assembly of signaling proteins. Features such as multivalency and intrinsic disorder that enable condensate formation are found not only in cytosolic and nuclear proteins, but also in membrane-associated proteins. The ABC transporter Rv1747, which is important for Mycobacterium tuberculosis (Mtb) growth in infected hosts, has a cytoplasmic regulatory module consisting of 2 phosphothreonine-binding Forkhead-associated domains joined by an intrinsically disordered linker with multiple phospho-acceptor threonines. Here we demonstrate that the regulatory modules of Rv1747 and its homolog in Mycobacterium smegmatis form liquid-like condensates as a function of concentration and phosphorylation. The serine/threonine kinases and sole phosphatase of Mtb tune phosphorylation-enhanced phase separation and differentially colocalize with the resulting condensates. The Rv1747 regulatory module also phase-separates on supported lipid bilayers and forms dynamic foci when expressed heterologously in live yeast and M. smegmatis cells. Consistent with these observations, single-molecule localization microscopy reveals that the endogenous Mtb transporter forms higher-order clusters within the Mycobacterium membrane. Collectively, these data suggest a key role for phase separation in the function of these mycobacterial ABC transporters and their regulation via intracellular signaling.

Keywords: ABC transporter; FHA domain; nanoclustering; phase separation.

Fig. 1.

Rv1747^1–310 undergoes phosphorylation-enhanced phase separation into liquid-like droplets in vitro. (A) Schematic representation of Rv1747 as a homodimer with the regulatory module (FHA domains, blue; reported PknF phospho-acceptor sites T152/T210 in the ID linker, red) appended to the core ABC transporter (transmembrane domain [TMD] and nucleotide binding domain [NBD], brown, with boundaries indicated). (B) Unmodified and phosphorylated Rv1747^1–310 phase-separated at different threshold concentrations. Shown are fluorescent images of unmodified (Top) and phosphorylated (Bottom) OG-Rv1747^1–310, taken at 120 min after removal from the concentration device or after the addition of 0.5 μM PknF and 100 μM ATP, respectively. (Scale bars: 40 μm.) (C) The indicated saturation concentrations for phase separation were quantified by fractional fluorescence intensity.

Fig. 2.

Diffusive exchange of phase-separated Rv1747^1–310 slows on phosphorylation. (A) FRAP measurements of exchange at room temperature in droplets formed by unmodified OG-Rv1747^1–310 (>250 μM; Top) or phosphorylated OG-Rv1747^1–310 (50 μM treated with 0.5 μM PknF and 100 μM ATP; Bottom). The bleached sector at t = 0 is indicated by an arrow, followed by images obtained at 2 subsequent time points. (Scale bars: 5 μm.) Although fluorescence recovery is likely dominated by diffusive exchange within the droplet, exchange with surrounding liquid phase is also possible. (B) FRAP recovery was quantified by fitting the average normalized intensity (solid dots with SD bars) to a single exponential function (black lines) for 3 different droplets in 1 sample of either unmodified or phosphorylated Rv1747^1–310.

Fig. 3.

Rv1747 phase separation is regulated by the Mtb STPKs and phosphatase. (A) PstP dissolved PknF-induced Rv1747^1–310 droplets. Condensates of 50 μM OG-Rv1747^1–310 were formed at room temperature in buffer containing 100 μM ATP and 0.5 μM PknF. After 120 min, the PstP phosphatase domain (5 μM) was added to 1 of 2 samples, and fluorescent images were recorded at the indicated time points. (Scale bars: 20 μm.) Dissolution occurred slowly, likely due to the competing activity of PknF with ATP still present in the sample and the requirement for only substoichiometric phosphorylation to induce cooperative phase separation. (B) Fluorescence images of OG-Rv1747^1–310 droplets induced by 5 Mtb STPKs (0.5–2 μM kinase domain plus 100 μM ATP) (Scale bars: 20 μm.) With a starting concentration of 10 μM, any unmodified Rv1747^1–310 was below its threshold concentration for phase separation. (See also SI Appendix, Fig. S10.) (C) The PknF and PstP catalytic domains colocalized to OG-Rv1747^1–310 droplets. After formation with nonlabeled PknF (0.5 μM with 100 μM ATP), 0.05 μM AF647-PknF (Top) or AF647-PstP (Bottom) was added. Images of OG (green) and AF647 (red) fluorescence were overlaid to monitor colocalization. (Scale bars: 2 μm.)

Fig. 4.

Rv1747^1–310 undergoes spontaneous clustering on supported lipid bilayers and in vivo. (A) Fluorescence images of nonphosphorylated His₆-tagged OG-Rv1747^1–310 anchored to supported lipid bilayers containing DGS-NTA(Ni²⁺). (Scale bars: 8 μm.) Phase separation was quantified by the fractional fluorescence intensity vs. weight percentage of the NTA(Ni²⁺) lipid, which sets the pseudo-2D concentration of anchored Rv1747^1–310. At higher DGS-NTA(Ni²⁺) levels, a characteristic spinodal decomposition (9) of the membrane-anchored droplets was seen. (B) Fluorescent images of yeast cells expressing msfGFP or msfGFP-Rv1747^1–310. Arrowheads indicate dense foci of msfGFP-Rv1747^1–310, and the arrow designates increased fluorescence intensities at the bud neck. Foci formation coincided with greater expression levels of msfGFP-Rv1747^1–310, likely driven by a higher plasmid copy number in these cells. Bud neck localization may be due to binding of the Rv1747^1–310 FHA domains to similar targets recognized by the endogenous FHA-containing protein Rad53 (33). (C) Representative total internal reflection fluorescence (TIRF) micrographs of live M. smegmatis expressing the indicated proteins. (Scale bars: 5 μm.) (Top Left) Raw images from the first frame of Movie S2 were analyzed using Icy Bioimaging particle detection software (Methods). (Bottom Left) Foci above the detection criteria are shown in the binary image output of the software. (Right) Kymographs illustrating the time evolution of fluorescence signals across the length of the bacterium in the raw image.

Fig. 5.

Nanoclusters of Rv1747 in Mtb. (A) Rv1747-specific AF647-scFv stained cell wall-labeled (green) Mtb, but not Δrv1747 Erdman (SI Appendix, Fig. S14). (B) Representative TIRF micrographs of GFP (Left) and anti-Rv1747 AF647 (Middle) from a single Mtb cell acquired before superresolution imaging. (Right) SMLM reconstructions of anti-Rv1747 AF647-scFv localizations, depicted as a color-coded density plot. (Scale bar: 500 nm.) (C) Single-molecule localizations were clustered in 3D using DBSCAN (32). The depicted clusters are color-coded by area. Each cluster contains at least 6 localizations (MinPts) within 30 nm (Eps) of each other. (D) Distribution function showing the cumulative fraction of all localizations from 45 Mtb cells. The y-intercept is the fraction of nonclustered localizations. Close to one-half (47%) of the localizations were organized into clusters larger than 2,600 nm², the estimated maximal area occupied by an Rv1747 molecule. (E) Model for phosphorylation-enhanced clustering and regulation of Rv1747. The transporter (core ABC structure, brown) clusters due to the concentration-dependent phase separation of the regulatory module (FHA domains, blue). This may increase the transporter activity (green arrow) of Rv1747 via an undefined mechanism. Phosphorylation (red circles) by Mtb STPKs reduces the saturation concentration for clustering (upper triangle). Additional interactions with regulatory proteins or transporter substrates could modulate the saturation concentration.