Genetic evidence for functional interaction of the Escherichia coli signal recognition particle receptor with acidic lipids in vivo

Abstract

The mechanism underlying the interaction of the Escherichia coli signal recognition particle receptor FtsY with the cytoplasmic membrane has been studied in detail. Recently, we proposed that FtsY requires functional interaction with inner membrane lipids at a late stage of the signal recognition particle pathway. In addition, an essential lipid-binding α-helix was identified in FtsY of various origins. Theoretical considerations and in vitro studies have suggested that it interacts with acidic lipids, but this notion is not yet fully supported by in vivo experimental evidence. Here, we present an unbiased genetic clue, obtained by serendipity, supporting the involvement of acidic lipids. Utilizing a dominant negative mutant of FtsY (termed NG), which is defective in its functional interaction with lipids, we screened for E. coli genes that suppress the negative dominant phenotype. In addition to several unrelated phenotype-suppressor genes, we identified pgsA, which encodes the enzyme phosphatidylglycerophosphate synthase (PgsA). PgsA is an integral membrane protein that catalyzes the committed step to acidic phospholipid synthesis, and we show that its overexpression increases the contents of cardiolipin and phosphatidylglycerol. Remarkably, expression of PgsA also stabilizes NG and restores its biological function. Collectively, our results strongly support the notion that FtsY functionally interacts with acidic lipids.

FIGURE 1.

FtsY relieves the toxic effect of NG. A, E. coli cells harboring pCV3-araP-NG were co-transformed either with plain vector (vec) or pT75-tacP-FtsY (FtsY). The transformants were plated on LB agar without (as a control) or with 0.5% arabinose for NG induction. B, overnight cultures were diluted and grown in LB broth. At A₆₀₀ (OD₆₀₀) of 0.03, the cultures were induced with 1% arabinose and grown for additional 4 h. Growth was followed by measuring the optical density of the cultures.

FIGURE 2.

Deletion and functional analysis of clones harboring the pgsA gene. A, schematic representation of several deletion constructs that were characterized. B, plasmids harboring the deletion constructs shown in A, containing 6 histidine tags at the C terminus of PgsA, were transformed into E. coli Top10/pT7-5-tacP-NG and examined for their ability to suppress the NG toxicity. C, overnight cultures of cells co-expressing NG and either plain vector (NG) or PgsA (NG+pgsA(+tRNAs)) were diluted and grown in LB broth. At A₆₀₀ (OD₆₀₀) of 0.03, the cultures were induced with 0.2 mm IPTG and grown for additional 5 h. Growth was followed by measuring the optical density of the cultures. D, Aska clone JW1897 (25) harboring the His₆-pgsA gene under the lac promoter (lacP) (without the tRNAs operon) was transformed into E. coli Top10/pCV-5-araP-NG and plated on LB agar plates with 0.5% arabinose and without IPTG, the PgsA inducer. E, E. coli cells co-expressing NG and either plain vector (vec), PgsA-His₆, PgsA-His₆ (+tRNAs), or lacP-His₆-PgsA (Aska clone JW1897) were grown in LB broth. Membrane fractions were isolated, and protein expression was analyzed by Western blotting using India HisProbe-HRP (for His₆-tagged PgsA identification). His₆-PgsA expressed under the regulation of lacP is larger than PgsA-His₆ expressed under its endogenous promoter due to addition of 15 amino acids derived from insertion of SfiI restriction sites (25).

FIGURE 3.

Effect of PgsA overexpression on lipid composition in vivo. A, thin layer chromatography separation of phospholipid standards. B, E. coli Top10 harboring plasmid-encoded variants of PgsA or empty vectors were labeled with [³²P]phosphoric acid at a final concentration of 1 μCi/ml and grown for 3 h at 37 °C. Lipids were extracted and separated by thin layer chromatography. C, quantitation of the phospholipid composition (moi % of lipid phosphorus calculated from radioactivity of each spot, see “Experimental Procedures”). The data shown are mean values, and the standard deviations are calculated from two or three different batches of cultures.

FIGURE 4.

Inactive PgsA mutant does not suppress NG toxicity. A, inactive PgsA mutant (T60P) was examined for its ability to suppress the NG toxic effect as in Fig. 2. vec, vector. B, E. coli co-expressing NG and either plain vector, PgsA-His₆ (+tRNAs) or PgsA(T60P)-His₆(+tRNAs) were grown in LB broth. Membrane fractions were isolated, and protein expression was analyzed by Western blotting using antibodies against FtsY for NG identification (left panel) or India HisProbe-HRP for PgsA identification (right panel).

FIGURE 5.

NG activation by PgsA. A, E. coli IY28-pT75-tacP-NG cells were transformed either with a plain vector (NG) or PgsA (NG+pgsA(+tRNAs)) and plated with (as a control) or without arabinose, the FtsY inducer. B, overnight cultures were grown with arabinose, washed four times, and diluted in LB broth without arabinose. Growth was followed by measuring the optical density of the cultures.

FIGURE 6.

PgsA expression stabilizes NG and restores membrane localization. A, E. coli BW25113 cells expressing NG alone (NG) or with PgsA (NG+ pgsA(+tRNAs)) were induced using 0.1 or 0.3 mm IPTG for 2 h. B, cells were induced using 0.5 mm IPTG for 30 min, rinsed four times to remove the inducer, and grown again. Samples were taken at different time points, and cells were fractionated. NG expression was analyzed by Western blotting using antibodies against FtsY. C, amount of NG was quantified by densitometry, and the average of three independent experiments is shown, with error bars representing standard deviations. D, flotation experiments with anionic phospholipids. In presence of zwitterionic phospholipids (phosphatidylethanolamine/phosphatidylcholine ratio of 70:30), no interaction was seen. Lipid interaction could be restored with increasing amounts of anionic phospholipids (10–50% phosphatidylglycerol). The vesicle fraction is marked by “v” and the pellet fraction by “p.” POPE, palmitoyloleoylphosphatidylethanolamine; POPG, palmitoyloleoylphosphatidylglycerol; POPC, palmitoyloleoylphosphatidylcholine.