Analysis of chloroquine resistance transporter (CRT) isoforms and orthologues in S. cerevisiae yeast

Abstract

Previous work from our laboratory optimized MeOH-inducible expression of the P. falciparum malarial parasite transporter PfCRT in P. pastoris yeast. These strains are useful for many experiments but do not allow for inducible protein expression under ambient growth conditions. We have therefore optimized galactose-inducible expression of PfCRT in S. cerevisiae yeast. We find that expression of PfCRT confers CQ hypersensitivity to growing yeast and that this is due to plasma membrane localization of the transporter. We use quantitative analyses of growth rates to compare hypersensitivity for yeast expressing various PfCRT isoforms. We also report successful high level inducible expression of the P. vivax orthologue, PvCRT, and compare CQ hypersensitivity for PvCRT vs PfCRT expressing yeast. We test the hypothesis that hypersensitivity is due to increased transport of CQ into yeast expressing the transporters via direct (3)H-CQ transport experiments and analyze the effect that membrane potential has on transport. The data suggest important new tools for rapid functional screening of PfCRT and PvCRT isoforms and provide further evidence for a model wherein membrane potential promotes charged CQ transport by PfCRT. Data also support our previous conclusion that wild type PfCRT is capable of CQ transport and provide a basis for understanding the lack of correspondence between PvCRT mutations and resistance to CQ in the important malarial parasite P. vivax.

FIG. 1. Western Blot analysis of gal – inducible CRT protein expression

A: Crude membrane preparations of S. cerevisiae transformed with different isoforms of PfCRT grown under non-inducing glucose conditions (lanes 1, 3, 5, 7) vs inducing galactose / raffinose (lanes 2, 4, 6, 8). Each lane contains 10 μg of protein. Each blot uses crude yeast membranes (total membrane fraction) prepared from CH1305 strains transformed with pYES2/PfHB3vh (Lanes 1 and 2), pYES2/PfDd2vh (Lane 3 and 4), pYES2/Pf7G8vh (Lane 5 and 6) or pYES2/PfHB3_PMAvh (Lanes 7 and 8). The V5-tagged proteins were detected with anti-V5-HRP. Top arrow (dashed) points to PfHB3_PMAvh protein, bottom (solid) arrow points to normal length PfCRT proteins. B: Crude membranes were prepared from CH1305 strains transformed with pYES2/PfHB3vh (Lane 1), pYES2/PfDd2vh (Lane 2), pYES2/Pf7G8vh (Lane 3), pYES2/PvCRTvh (Lane 4) or pYES2/PvCRT_Dd2vh (Lane 5) and equivalent membrane protein (quantified by amido black assay) run side – by – side. Top panel shows anti-pentaHis staining, bottom shows anti-V5 staining, both of which indicate that approximately equivalent levels of the isoforms and orthologues are expressed in the different strains. C: Comparison of relative amounts of PfHB3vh and pYES2/PfHB3_PMAvh in plasma membrane fractions. Lanes 1 and 5 are 5 μg of crude membranes prepared from CH1305 strains transformed with pYES2/PfHB3vh and pYES2/ PfHB3_PMAvh. Lanes 2 and 6 are 5 μg of pellet harvested at 75, 000 × g (intermediate step in pure plasma membrane preparation, harboring non – plasma membrane cellular membranes) from CH1305 strains transformed with pYES2/PfHB3vh and pYES2/PfHB3_PMAvh. Lanes 3, 4 and 7, 8 contain 5 and 10 μg, respectively, of pure plasma membranes harvested from CH1305 strains transformed with pYES2/PfHB3vh and pYES2/ PfHB3_PMAvh. Top arrow indicates PMA1-fusion PfHB3 isoform and bottom arrow indicates PfHB3 isoform. High mass bands near 100 kDa in lanes 7,8 are likely PMA1 – HB3CRT aggregates, and band near 15 kDa is an apparent protease degradation product. Densitometry of bands indicated approximately 50 – 60 percent of CRT is within the plasma membrane fraction without N terminal modification, but for pYES2/ PfHB3_PMAvh, > 10 times is found in the plasma membrane relative to all other cell membranes.

FIG. 2. Plate Assays for CQ and CRT – dependent growth inhibition

Susceptibilities of CH1305 yeast harboring pYES2 (“empty vector” or “EV”), pYES2/LacZ, pYES2/PfHB3vh, pYES2/PfDd2vh, pYES2/Pf7G8vh, pYES2/PvCRTvh, pYES2/PvDd2vh or pYES2/PfHB3_PMAvh. Suspensions for inoculating plates were grown at 30°C to mid-exponential phase in SD media - uracil. Suspensions equilibrated to OD = 0.1 were transferred to 96 well plates, 10-fold serial dilutions were made, and 5 uls of each (10⁴_, 10³, 10² cells; top, middle and bottom row, respectively, each “box” defined by white dashed lines) was then replica plated on SD or SGR media – uracil, +/− concentrations CQ. Plates were incubated side-by-side for approximately 3 days at 30°C.

FIG. 3. AzBCQ Photolabelling of Partially Purified PvCRT

A: Representative streptavidin-HRP detection of AzBCQ photolabelled PvCRT vs. competition with underivitized CQ (see [9] and [13] for detailed description of methods). Lane 1: no CQ competitor, Lane 2-6: 5-, 10-, 20-, 40-, 80-fold molar excess of CQ relative to AzBCQ, respectively. B: PentaHis-HRP detection of PvCRT in the same samples as in A, showing equal amounts of protein were used in each photolabelling reaction (see [13]). C: Plot of densitometry of bands as in A, vs. [CQ] competitor. As described [13], results from 3 different experiments (three photolabelling reactions, three separate sets of gels) were averaged, data represent mean +/− S.E.M. Fitting this curve shows that 50 % maximal inhibition of PvCRT CQ analogue photolabelling occurs at 8.5 (+/− 1.3) – fold molar excess of underivitized CQ.

FIG. 4. Quantitative growth curve analysis

Susceptibilities of yeast strain CH1305 harboring pYES2 (Empty vector, top), pYES2/PfHB3vh, pYES2/PfDd2vh, pYES2/Pf7G8vh or pYES2/PvCRTvh were assessed by quantitative growth curve analysis in liquid medium, pH = 5.0. Cultures were inoculated with 10⁵ cells/ml synthetic complete medium with (A, left side panels) 3% glucose (SD-ura) alone (solid lines) or with 100 mM CQ (dashed), or (B, right side panels) synthetic complete medium with 2% galactose and 1% raffinose (SGR-ura) alone (solid), or SGR-ura with 100 mM CQ (dashed). Average growth curves from three replicates are shown, see Fig. 5 for curve fit analysis.

FIG. 5. Summary of Quantitative Growth Analysis

Doubling rates for data obtained as in Fig. 4 were computed as described. A: Inducing SGR medium, pH = 5.0 with (gray bars) or without (black bars) 100 mM CQ, B: Inducing SGR medium, buffered to pH = 7, with (gray bars) or without (black bars) 75 mM CQ. C: Induction and pH / ΔΨ-dependent effects on growth rate. Growth rates obtained for non-inducing medium + 75 mM CQ were subtracted from rates obtained for inducing medium + 75 mM CQ. The growth rate differences for yeast expressing Dd2 PfCRT (top, triangles) were significantly higher relative to yeast expressing HB3 PfCRT (middle, squares) for all pH / ΔΨ (P < .05, unpaired t tests in each comparison). As expected, growth under the two conditions did not differ significantly for yeast harboring empty vector (bottom, diamonds). The data represent the means (+ S.E.M) of 4 independent experiments.

FIG. 6. ³H-CQ accumulation in yeast +/− CRT expression

CH1305 harboring pYES2 (closed circles) or pYES2/PfDd2vh (open circles) were grown to exponential phase in SGR/-ura, harvested and assayed for [³H]-CQ uptake at pH 5.5 (A) or 7.5 (B). The data represent the means (± S.D.) for 3 - 5 separate experiments.

FIG. 7. Summary of ³H – CQ accumulation data for different yeast strains

CH1305 harboring pYES2 (“EV”), pYES2/PfHB3vh, pYES2/PfDd2vh, pYES2/Pf7G8vh or pYES2/PvCRT were grown to exponential phase in SGR/-ura, harvested and assayed for [³ H] CQ uptake at 30 min (near plateau, see Fig. 6) at either pH 5.5 (black bars) or pH 7.5 (gray bars). Similar results are obtained at 60 min time (not shown, see Fig. 6). Each bar represents the average of 3 separate experiments. *P > .2, ** P=.08, *** P<.01. Differences in uptake at pH 7.5 vs. 5.5 are significant for 7G8 (P < .05, unpaired t tests) and suggestive for Dd2 (P = .08, unpaired t tests), but not significant for any of the other strains.

FIG. 8. Initial rate analysis of ³H – CQ accumulation

Representative initial rate analysis of ³H – CQ accumulation (see also Table 3 for a summary). Gray squares are CH1305 expressing pYES2/PfDd2vh, black diamonds CH1305 expressing pYES2. Data were obtained at pH 5.5 and are the results of three experiments, +/− S.D. See Table 3 for a summary of initial rate data.