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. 2007 Dec 4;104(49):19500-5.
doi: 10.1073/pnas.0708818104. Epub 2007 Nov 29.

NIX is required for programmed mitochondrial clearance during reticulocyte maturation

Rachel L Schweers  1 , Ji Zhang, Mindy S Randall, Melanie R Loyd, Weimin Li, Frank C Dorsey, Mondira Kundu, Joseph T Opferman, John L Cleveland, Jeffery L Miller, Paul A Ney
Affiliations

NIX is required for programmed mitochondrial clearance during reticulocyte maturation

Rachel L Schweers et al. Proc Natl Acad Sci U S A. .

Abstract

The regulated clearance of mitochondria is a well recognized but poorly understood aspect of cellular homeostasis, and defects in this process have been linked to aging, degenerative diseases, and cancer. Mitochondria are recycled through an autophagy-related process, and reticulocytes, which completely eliminate their mitochondria during maturation, provide a physiological model to study this phenomenon. Here, we show that mitochondrial clearance in reticulocytes requires the BCL2-related protein NIX (BNIP3L). Mitochondrial clearance does not require BAX, BAK, BCL-X(L), BIM, or PUMA, indicating that NIX does not function through established proapoptotic pathways. Similarly, NIX is not required for the induction of autophagy during terminal erythroid differentiation. NIX is required for the selective elimination of mitochondria, however, because mitochondrial clearance, in the absence of NIX, is arrested at the stage of mitochondrial incorporation into autophagosomes and autophagosome maturation. These results yield insight into the mechanism of mitochondrial clearance in higher eukaryotes. Furthermore, they show a BAX- and BAK-independent role for a BCL2-related protein in development.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Expression of BCL2-related proteins during terminal erythroid differentiation. Expression of NIX, BCL-XL, and other BCL2-related proteins during FVA cell differentiation. Wild-type and Nix−/− FVA cells were cultured for 48 h in the presence of erythropoietin. NIX monomer and a lower molecular mass form of NIX (asterisk) are shown. Molecular mass marks are shown on the right.
Fig. 2.
Fig. 2.
Defective mitochondrial clearance in Nix−/− reticulocytes. (A) Blood smears from Nix+/+ and Nix−/− mice. The Nix−/− blood smear shows polychromatophils (blue-tinged cells), spherocytes (absent central pallor), and abnormally shaped erythrocytes. The slides are stained with Wright–Giemsa. (B) Flow cytometry of Nix+/+ and Nix−/− blood stained with TO. The asterisk marks erythrocytes that failed to clear their mitochondria. (C–F) Ultrastructure of erythrocytes from the blood of Nix+/+ (C) and Nix−/− (D–F) mice. Nix−/− erythrocytes contain mitochondria and ribosomes (D) and double-membraned autophagosomes (E and F, arrows). (G) Flow cytometry of wild Nix+/+ and Nix−/− blood stained with MTR. (H) Flow cytometry of erythrocytes stained with TO and MTR. (I) Flow cytometry of Nix+/+ and Nix−/− reticulocytes stained with MTR at baseline (black lines) or after (gray shading) 3 days in culture. (J) Immunofluorescence imaging of cultured Nix+/+ and Nix−/− reticulocytes stained with MTR.
Fig. 3.
Fig. 3.
Mitochondrial clearance is BAX-, BAK-, and BCL-XL-independent. (A and B) Flow cytometry of erythrocytes stained with TO and MTR. Genotypes are indicated. (C) Western blots of BCL-XL, BAX, BAK, and succinate dehydrogenase subunit B (SDHB). Reticulocytes were obtained from wild-type and BclXfl/fl;Bax−/fl;Bak−/−;Tg(MMTV-Cre) (triple mutant) phlebotomized mice by positive selection for CD71 expression.
Fig. 4.
Fig. 4.
Ubiquitin-like conjugation activity is induced independently of NIX. Nix+/+ and Nix−/− FVA cells were cultured for 60 h in the presence of EPO. LC3-I is the unconjugated form of LC3, and LC3-II is the lipid-conjugated form of LC3. Beclin-1 levels are shown for comparison. Wild-type LC3-overexpressing murine embryonic fibroblasts untreated (UN) or treated with chloroquine (CQ), which causes accumulation of the lipid-modified LC3 (F.C.D. and J.L.C., unpublished results), were used to confirm the identity of LC3-I and LC3-II.
Fig. 5.
Fig. 5.
Mitochondrial autophagy is defective in Nix−/− reticulocytes. Ultrastructure of Nix+/+ (Upper) and Nix−/− (Lower) reticulocytes. (Upper) Regular white arrows point to individual mitochondria in vacuoles, and closed white arrows point to mitochondria in mature autophagic vacuoles. Both were observed undergoing exocytosis. (Lower) Barbed white arrows point to isolation membranes and double-membraned autophagosomes. The length of time in culture (days) is indicated in the lower right corner of each panel as d0, d1, d2, or d3.
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