We used a Hybrid Mouse Diversity Panel comprised of 100 inbred mouse strains8
to identify Abcc6
gene networks. We found that liver transcripts whose expression significantly correlated with Abcc6
>0.390) were highly enriched for mitochondrial genes (Online Table I). Likewise, when we compared liver and kidney transcripts from mice with nonfunctional ABCC6 versus functional ABCC6 we, again, found an enrichment for mitochondrial genes (Online Table II). We therefore investigated the subcellular location of the protein. We compared the C3H inbred mouse strain, which has an endogenous Abcc6
splice site mutation resulting in reduced transcripts and nonfunctional proteins,9
to C3H Abcc6
transgenic mice (Abcc6
-Tg) and C57BL/6 Abcc6
-wildtype (WT) to Abcc6
-KO mice on the C57BL/6 background (Online Table III). Both C3H and Abcc6
-KO mice are Abcc6
-deficient and have extensive vascular calcification and other pathologies resembling PXE,4
which are rescued in Abcc6
We first conducted subcellular fractionation using a sucrose gradient and observed strong ABCC6 signals in immunoblots of mitochondrial fractions in both the liver () and kidney (Online Figure I) of Abcc6
-Tg mice. ABCC6 signals were lower in C3H as expected. The mitochondrial isolation was validated by immunoblotting for compartment-specific markers (). Most proteins were enriched in their known subcellular location; however, the endoplasmic reticulum (ER) marker, protein disulfide isomerase, was also present in the mitochondrial fraction, suggesting further purification was needed to eliminate the possibility that ABCC6 was an ER protein. Before proceeding, we confirmed the specificity of the ABCC6/MRP6 (S-20) antibody.10
In our experiments, the antibody recognized a single predominant band of ≈160 kDa. The signal intensity was highest for Abcc6
-Tg, present in WT, significantly lower in C3H, and absent in Abcc6
-KO mice (). Moreover, the signal in Abcc6
+/− heterozygotes was approximately half of that in WT (Online Figure II). The immunoreactive ABCC6 protein was N-glycosylated (Online Figure III), characteristic of full-length ABCC6 expressed in mammalian cells.11
We therefore concluded that the ≈160-kDa band in the mitochondrial fraction corresponded to ABCC6.
ABCC6 (ATP-binding cassette, subfamily C, member 6) localization in the mitochondrial fraction following subcellular fractionation
To corroborate the subcellular fractionation findings, we investigated purified mitochondria using confocal microscopy. The isolated kidney crude mitochondrial fraction was Percoll-purified to reduce microsomal contamination. ABCC6 immunofluorescence showed clear colocalization with functional mitochondria from WT kidney (). The specificity of immunofluorescence was confirmed by the absence of ABCC6 signal in Abcc6-KO mitochondria. Notably, ABCC6 was detected in a subset of mitochondria, as opposed to the respiratory chain complex subunit COXIV (Online Figure IV). In ultrahigh resolution images, ABCC6 was visible as distinct clusters of ≈20 and ≈40 nm ().
The detection of ABCC6 in a subset of mitochondria could be due to Abcc6
expression in specific cell types or in a subpopulation of cellular mitochondria. To address the latter possibility, we examined the MAM, a specialized cellular compartment that bridges the ER with some but not all mitochondria in the cell.12
The MAM and mitochondrial outer membrane couple through protein-protein interactions but constitute separate lipid layers. Because the MAM was not completely removed in our immunofluorescence experiment, we further separated the MAM and other microsomal contamination by ultracentrifugation.7
Immunoblotting of the resulting fractions revealed that ABCC6 resides in the MAM fraction (). This fraction contained minimal to no contamination from mitochondria, cytosol, and caveolae. Remarkably, ABCC6 was more enriched than known MAM markers including calnexin.7
ABCC6 was undetectable in the highly purified mitochondrial fraction and only faintly visible in pure non-MAM ER.
ABCC6 (ATP-binding cassette, subfamily C, member 6) localizes to mitochondria-associated membrane (MAM) in mice
Previous studies using immunohistochemical staining have suggested that ABCC6 localizes to the plasma membrane.1,10
To rule out the possibility of dual ABCC6 localization to MAM and plasma membrane, we biotin-labeled cell surface proteins of primary hepatocytes from WT mice. Following affinity precipitation with streptavidin, the cell surface marker pan-cadherin was retained, but not ABCC6, which associated entirely with the intracellular fraction (). The result indicates the majority of ABCC6 protein is intracellular.
We previously showed that C3H and Abcc6
-KO mice suffer increased cardiac necrosis following cardiac ischemia-reperfusion.13
To investigate the possible mitochondrial contribution to this result, we used electron microscopy to image liver, kidney, and heart tissue. Liver electron microscopy sections did not reveal mitochondrial differences but showed a reduction in ER bordering the mitochondria (). Most mitochondria in Abcc6
-KO liver were surrounded by a single strand of ER, but the extensive ER clusters between mitochondria, present in the WT, were absent (Online Figure V). In the Abcc6
-KO kidney proximal tubule, >40% of mitochondria had virtually no cristae ( and Online Figure VI), compared to ≈10% mitochondria in WT. Heart cross-sections displayed striking abnormalities of the cristae (), along with inconsistent size and numbers of mitochondria per sarcomere (Online Figure VII).
Abcc6 (ATP-binding cassette, subfamily C, member 6) deletion associates with mitochondrial dysfunction and dysmorphology
The morphological defects in Abcc6-KO kidney and heart prompted us to measure mitochondrial respiration. Normal mitochondria responded to the uncoupler FCCP with a sharp increase in oxygen consumption rate (respiratory reserve), whereas Abcc6-KO liver, kidney, and heart mitochondria all demonstrated significantly lower response on uncoupling (). These findings corroborate the mitochondrial dysmorphology in the kidney and heart and suggest functional defects in hepatic mitochondria despite inapparent structural abnormalities.
Our microarray results suggested that of the known MAM functions, protein processing and purine nucleotide activity were the most impacted by ABCC6 deficiency (Online Table IV). Calcification pathway proteins, protein C, ENPP1, and GGCX, a key protein involved in vitamin K-mediated calcification, were differentially expressed.