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. 2019 Jun 4;8:e43923. doi: 10.7554/eLife.43923

Figure 4. NPC2 binding to symbiont-produced sterols via immunoprecipitation-lipidomics, and differential stability of non-canonical NPC2 at varying pH.

(A) Quantification of bound lipids in the eluates following IP of recombinant canonical and non-canonical NPC2:crmCherry:HA and negative control crmCherry:HA at pH 7 (Figure 4—figure supplement 1). Sterols comprising <1.5% were omitted for clarity. crmCherry, lysosome-stable cleavage-resistant mCherry (Huang et al., 2014) . Average values ± SD (error bars). Except ß-sitosterol, each NPC2 was significantly different to crmCherry negative control (Student’s t-test, p<0.01). (B) Relative proportions of NPC2-bound sterols and the corresponding symbiont extract at pH 5 and 7. Average values ± SD (error bars) (C) Immunoprecipitation (IP) of native non-canonical NPC2 from Aiptasia and quantification of eluted bound sterols. Control, identical reaction omitting antibody. Average values ± SEM (error bars). Statistical comparisons to control (Student’s t-test: *p<0.05, •p<0.09). (D) NPC2 gene expression by qPCR in the various Aiptasia/Symbiodiniaceae host/symbiont combinations in Figure 1B. (E) Recombinant NPC2s detected by mCherry antibody in the soluble fractions of HEK cell lysates. Lysate preparations were identical except for buffer pH; equivalent volumes loaded per lane. (F) Quantification of protein abundances from Western blots in E. Average values ± SEM (error bars). Student’s t-test, *p<0.05. (G) We propose a model in which symbiotic anthozoans have evolved non-canonical NPC2 homologues that are spatiotemporally regulated to specifically respond to symbiosis, including through adaptation to the acidic environment of the symbiosome, the lysosomal-like organelle in which symbionts reside. NPC2 proteins bind and transport symbiont-produced sterols, and such trafficking is essential for cellular homeostasis of the sterol-auxotrophic hosts.

Figure 4.

Figure 4—figure supplement 1. Immunoprecipitation-lipidomics: protein inputs and immunoprecipitation (IP) elutions.

Figure 4—figure supplement 1.

HEK cell lysate input containing recombinant NPC2s or crmCherry control, and subsequent protein elutions of anti-HA IP reactions, in duplicate. After washing, lipids were eluted (Figure 4A + B) from IP reactions prior to protein elution shown here.

Figure 4—figure supplement 2. Soluble recombinant NPC2s in HEK 293T cell lysates.

Figure 4—figure supplement 2.

Full Western blots from which the sections are shown in Figure 4E.