A brain-enriched Drp1 isoform associates with lysosomes, late endosomes, and the plasma membrane

Kie Itoh,Yoshihiro Adachi,Tatsuya Yamada,Takamichi L Suzuki,Takanobu Otomo,Heidi M Mcbride,Tamotsu Yoshimori,Miho Iijima,Hiromi Sesaki

doi:10.1074/jbc.ra117.001253

Abstract

Dynamin-related protein 1 (Drp1) constricts mitochondria as a mechanochemical GTPase during mitochondrial division. The Drp1 gene contains several alternative exons and produces multiple isoforms through RNA splicing. Here we performed a systematic analysis of Drp1 transcripts in different mouse tissues and identified a previously uncharacterized isoform that is highly enriched in the brain. This Drp1 isoform is termed Drp1ABCD because it contains four alterative exons: A, B, C, and D. Remarkably, Drp1ABCD is located at lysosomes, late endosomes, and the plasma membrane in addition to mitochondria. Furthermore, Drp1ABCD is concentrated at the interorganelle interface between mitochondria and lysosomes/late endosomes. The localizations of Drp1ABCD at lysosomes, late endosomes, and the plasma membrane require two exons, A and B, that are present in the GTPase domain. Drp1ABCD assembles onto these membranes in a manner that is regulated by its oligomerization and GTP hydrolysis. Experiments using lysosomal inhibitors show that the association of Drp1ABCD with lysosomes/late endosomes depends on lysosomal pH but not their protease activities. Thus, Drp1 may connect mitochondria to endosomal-lysosomal pathways in addition to mitochondrial division.

Highlights

Dynamin-related protein 1 (Drp1) constricts mitochondria as a mechanochemical GTPase during mitochondrial division
We identified a new Drp1 isoform, Drp1ABCD, which predominantly expresses in the brain and locates at lysosomes, late endosomes and the plasma membrane in addition to mitochondria
The unique localization of Drp1ABCD to lysosomes, late endosomes, and the plasma membrane depends on the combination of exons A and B

Summary

Results

We systematically analyzed the expression profile of Drp transcripts using mRNAs isolated from different mouse organs and cells, including the brain, heart, skeletal muscle, liver, spleen, testis, lung, and embryonic fibroblasts. We noticed that Drp1ABCD, but not the other nine isoforms, is located at vesicular structures in the cytosol in addition to mitochondria when we changed focal planes (Fig. 2A, circled area) To characterize these vesicles, we performed confocal immunofluorescence microscopy with antibodies against Drp and Lamp, a membrane protein associated with lysosomes and late endosomes. We introduced the G350D mutation, which inhibits the oligomerization of Drp1 [25], and expressed Drp1ABCD (G350D) in Drp1-KO MEFs. Confocal immunofluorescence microscopy showed that Drp1ABCD (G350D) diffuses in the cytosol and does not associate with Lamp1-positive vesicles, the plasma membrane, or mitochondria (Fig. 7, C and D). These data suggest that the acidity of lysosomes, but not their function, is required for the association of Drp1ABCD with lysosomes/late endosomes

Discussion

Mice and MEFs

Western blotting

Mitochondrial respiration

Lentiviruses and plasmids

Immunofluorescence microscopy