Fig. 5: The N-terminus of ABHD10 is required for its mitochondrial localization.

a–c Schematic illustration of the targeting strategy for constructing ABHD10 protein HA-tagged mice. Due to the transit peptide is cleaved off during protein processing, HA tag was inserted after the transit peptide (b). Briefly, HA tag were inserted into the exon 2 of ABHD10 gene (a). After transcription and translation, ABHD10 HA tagged protein was produced (c). Images of mouse were generated by SciDraw (credit Ethan Tyler and Lex Kravitz), licensed under CC BY 4.0 (https://creativecommons.org/licenses/by/4.0/). d IHC staining using testicular tissue showed that ABHD10 was expressed in the testis and localized in the cytoplasm of germ cells and flagella of sperm. Scale bar: 20 μm. e WB analyses revealed that ABHD10 was expressed only in mitochondria of testes. Nc nuclei, Mito mitochondria, Cp cytoplasm (mitochondria has been removed). f Schematic for isolating mitochondrial subcellular component proteins. Mitochondria contains four sub-compartments: outer membrane (MOM), inner membrane (MIM) intermembrane space (IMS) and matrix. After treatment with sodium carbonate, mitochondria can be separated as pellet (PEL) containing integral IMM and integral OMM and supernatant (SN) containing Matrix, IMS, peripheral lMM and peripheral OMM. Image of mitochondria were generated by figdraw.com. g WB analyses of proteins in different sub-compartments of mitochondria. Mito Mitochondria, SN supernatant, PEL pellet. h Subcellular localization of ABHD10 in mouse testes using immunogold staining, followed by TEM. Representative image of mitochondria in testis showed that the specific gold particles localized in the matrix of mitochondria. Scale bar: 1 μm. i Super-resolution images of mitochondria in HEK293T cells. Green signal indicates GFP-ABHD10 fusion protein. IMM was labeled with PK mito (Purple signal). The white arrowheads indicate swollen mitochondria. Scale bar: 10 μm. j Subcellular localization of ABHD10 in HEK293T cells using immunogold staining, followed by TEM. The FLAG antibody was utilized to detect the expression of ABHD10 proteins. Representative image of mitochondria in HEK293T cells demonstrated that ABHD10 was localized in the matrix of mitochondria. Scale bar: 1 μm. k Diagrams showing the protein region organization of ABHD10 fused with GFP, including full length, the N-terminus deletion (ΔN), AB hydrolase-1 domain deletion (ΔD), and the C-terminus deletion (ΔC). l Representative images of 293T cells transfected with GFP vector or GFP-tagged ABHD10 full length or different deletions (ΔN, ΔD, and ΔC). The white arrowhead indicates that loss of mitochondrial co-localization of ABHD10 upon deletion of its N-terminus. Scale bar: 5 μm. d, e, g, h, i, j, l show representative images from at least three independent experiments with similar results.