Transmembrane Molecular Pump Activity of Niemann-Pick C1 Protein Joanna P. Davies, Fannie W. Chen, and Yiannis A. Ioannou

Supplementary Material

Supplemental Figure 1. Inhibition disk assay demonstrates the increased susceptibility of NPC1-expressing E. coli to acriflavine. The red lines indicate the inhibitory zone in uninduced E. coli, whereas the blue extensions of the line indicate the wider inhibitory zone exhibited by NPC1-expressing bacteria. Each disk contains the indicated amount of acriflavine (from left to right, 5, 10, and 20 mg). Plates were grown at 37 ° C for 16 hours.

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Supplemental Figure 2. Growth inhibition of NPC1-expressing E. coli by acriflavine. Control (-IPTG) and NPC1-expressing (+IPTG) E. coli cultures were grown in the presence of various concentrations of acriflavine for 30 min. Growth was assessed spectrophotometrically (_OD600).

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Supplemental Figure 3. Fractionation of E. coli membranes and detection of NPC1. Bacteria were lysed by sonication and crude lysates were clarified by centrifugation at 10,000g. The supernatant was subsequently subjected to ultracentrifugation at 100,000g to isolate bacterial membranes. NPC1 can be clearly seen in the purified membrane fraction. The lower panel is a magnified view of the relevant region shown on the gel.

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Supplemental Figure 4. (A) Isolation of an E. coli outer membrane permeability mutants.
Using an alkaline phosphatase secretion screen, a procedure described by Beja et al. (1), we isolated two E. coli permeability mutants. These "leaky" mutants secrete high levels of alkaline phosphatase, a periplasmic enzyme, indicating that their outer membrane is permeable. (B) Identification of permeability mutant 2 on X-phosphate plates showing the secretory phenotype (arrow).

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Supplemental Figure 5. Cholesterol and cholesterol-oleate uptake studies. Control and NPC1-expressing E. coli were incubated with [3H]cholesterol or [3H]cholesterol-oleate for 15 min. There were no detectable differences in lipid accumulation between control and NPC1-expressing cells.

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Supplemental Figure 6. Oleic acid transport by NPC1 across the E. coli plasma membrane. To establish the specificity of oleic acid transport by NPC1 protein, a second, related protein, NPC1L1(2), that shares a 52% amino acid homology with NPC1 and is predicted to also share a similar membrane topology with NPC1, was similarly expressed in E. coli. Oleic acid transport studies with NPC1- and NPC1L1-expressing bacteria demonstrate that only NPC1 transports oleic acid across the plasma membrane. The + or - signs indicate the presence or absence of the inducer IPTG, respectively. 2.1.1 indicates parental control cells.

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References

1. O. Beja, E. Bibi, Proc. Natl. Acad. Sci. U.S.A. 93, 5969 (1996).

2. J. P. Davies, B. Levy, Y. A. Ioannou, Genomics 65, 137 (2000).