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. 2017 Sep 5;114(38):E8045–E8052. doi: 10.1073/pnas.1700632114

Fig. 5.

Fig. 5.

CLPX and CLPXP assembly and models of the disease mechanism. (A) Coimmunoprecipitation of purified murine CLPX variants to assess heterooligomerization of WT and G298D variants. 3xFLAG-tagged ClpX was immunoprecipitated from a mixture with untagged CLPX or CLPXGD. The precipitate was separated by SDS/PAGE and stained with Sypro Orange. (B) Pull-down to assess the interaction of CLPX variants (WT or G298D) with CLPP-His6. 3xFLAG-tagged WT CLPX was used to distinguish it from CLPXGD. CLPX were incubated with CLPP-His6 and His-Tag Dynabeads in the presence or absence of 2.5 mM ATPγS. Coisolated proteins were separated by SDS/PAGE and stained with Sypro Red. With ATPγS, total pull-down of CLPX with CLPP in the mixed WT and G298D CLPX sample was 0.5 ± 0.1 relative to WT CLPX; no significant association of CLPXGD alone with CLPP was observed. P = 0.002 (Student’s t test, n of 3) for reduced pull-down of mixed WT CLPX and CLPXGD with CLPP. (C) Models of CLPX regulation of ALAS. (Left) Under normal circumstances, CLPX (blue circles) degrades ALAS in complex with CLPP (dark-blue cylinder), keeping ALA levels in check, preventing PPIX from accumulating (10). Concurrently, CLPX activates ALAS by promoting PLP incorporation (9). Note that direct degradation by CLPXP of ALAS1 or ALAS2 has not been demonstrated in vitro, and the influence of CLPX on ALAS stability could be more complex. (Middle) In a CLPXWT/GD heterozygote, CLPXGD (red circles) heterooligomerizes with CLPX, to form a dominant inhibitor of the ATPase activity of the hexamer. The resultant CLPXP complex is deficient in proteolytic activity, leading to an accumulation of ALAS that is more severe than any deficiency in PLP incorporation. (Any defect in ALAS activation by the heterohexameric CLPX has not been quantified, but likely is less impaired than in ALAS degradation; see text). This combination results in abnormally high ALAS activity and accumulation of PPIX, contributing to EPP. (Right) When CLPX is lost, reduced activation of ALAS is phenotypically stronger than increased ALAS protein, leading to decreased ALA production and anemia (9).