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To the Editor
In the course of studying the effects of ABCC6 deficiency in mice, we observed an enrichment of mitochondrial gene expression signatures. In subsequent studies, we found that ABCC6 null mice exhibited abnormal mitochondrial morphology and functional mitochondrial deficiencies.1 We then carried out subcellular fractionation studies, indicating that ABCC6 colocalized with markers of the mitochondria-associated membranes in mouse liver and kidney.1 Our results differed from Le Saux et al2 from the Varadi group, who had concluded in a recent publication that ABCC6 resided in the plasma membrane. To test whether ABCC6 was localized in plasma membrane, we performed cell surface protein biotin labeling experiments, which were negative for ABCC6.1
See Research Commentary, p e148
Pomozi et al3 of the Varadi group have now challenged our conclusions based, as in their previous report,2 on immunofluorescent imaging of frozen liver sections and cells in culture showing peripheral cellular localization of antibody binding. They argue that cell disruption and subcellular fractionation in our study1 may have resulted in artifactual associations of membrane proteins. However, this seems improbable, given that the plasma membrane markers that we examined did not fractionate with ABCC6, and mitochondria-associated membranes constitute a very small fraction of the total membranes.1 Cellular fractionation techniques have been used almost universally to provide definitive evidence of subcellular localization.4,5 Pomozi et al3 also argue that ABCC6, lacks sufficient amine groups on the extracellular surface to allow biotin labeling. This possibility cannot be excluded, although the manufacturer of the surface protein biotin labeling assay (Thermo Pierce) indicates that it is sensitive to even a single amine group, which would be present in the N terminus or in one of the predicted available lysines.
In our studies, we used the N-terminal binding (S-20) antibody from the same commercial supplier (Santa Cruz Biotechnology) as reported by Pomozi et al.3 In our hands, this antibody exhibited significant nonspecific binding in ABCC6 null mouse tissue sections (not shown) and in Western blots.1 We note that in their publication,2 the methods state that the S-20 antibody is made in rabbit and blocking was performed using goat serum. In fact, the antibody is made in goat and blocking with goat serum would produce nonspecific labeling (per Santa Cruz Biotechnology). An advantage of our results obtained by subcellular fractionation compared with immunofluorescence of tissue sections is that, in the former, the protein is detected on Western blots after separation by gel electrophoresis, thus allowing better separation from cross-reacting proteins. A lack of accompanying Western data in the current challenge, in our opinion, significantly weakens the ability to ascertain the specificity of the signal.
In conclusion, we recognize that localization of proteins can be challenging and is highly dependent on the quality of the antibody reagents. Although we cannot exclude the possibility that some of the ABCC6 protein resides on the plasma membrane, low-resolution imaging studies of frozen liver sections and cells in culture do not provide convincing evidence against the localization of ABCC6 in the mitochondria-associated membranes.
Lisa J. MartinDepartment of Medicine
Edward LauDepartments of Medicine and Physiology
Harpreet SinghDepartment of Anesthesiology
Laurent VergnesDepartment of Human Genetics
Elizabeth J. TarlingMargarete MehrabianImran MungrueSheila XiaoDiana ShihLawrence CastellaniDepartment of Medicine
Peipei PingDepartments of Medicine and Physiology
Karen ReueDepartment of Human GeneticsMolecular Biology Institute
Enrico StefaniDepartment of Anesthesiology
Thomas A. DrakeDepartment of Pathology and Laboratory Medicine
Kristina BostromDepartment of Medicine
Aldons J. LusisDepartments of Medicine, Human Genetics, andMicrobiology, Immunology and Molecular GeneticsDavid Geffen School of Medicine University of CaliforniaLos Angeles, CA

References

1.
Martin LJ, Lau E, Singh H, et al. ABCC6 localizes to the mitochondria-associated membrane. Circ Res. 2012;111:516–520.
2.
Le Saux O, Fülöp K, Yamaguchi Y, Iliás A, Szabó Z, Brampton CN, Pomozi V, Huszár K, Arányi T, Váradi A. Expression and in vivo rescue of human ABCC6 disease-causing mutants in mouse liver. PLoS One. 2011;6:e24738.
3.
Pomozi V, Le Saux O, Brampton CN, Apana ALY, Iliás A, Szeri F, Martin L, Monostory K, Paku S, Sarkadi B, Szakács G, Varadi A. ABCC6 is a basolateral plasma membrane protein. Circ Res. 2013;112. DOI: 10.1161/CIRCRESAHA.111.300194
4.
Huber LA, Pfaller K, Vietor I. Organelle proteomics: implications for subcellular fractionation in proteomics. Circ Res. 2003;92:962–968.
5.
Rockstroh M, Müller S, Jende C, Kerzhner A, von Bergen M, Tomm JM. Cell fractionation: an important tool for compartment proteomics. JIntegrated OMICS. 2011;1:135–143.

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Immunofluorescent staining of PiPS-SMCs for SM22 and DKK3. Partially-induced pluripotent stem cells were directly differentiated into smooth muscle cells (PiPS-SMCs). PiPS cells were differentiated for 4 days on collagen IV-coated plates in differentiation media. They were then subjected to immunofluorescent staining for SM22 and DKK3. DAPI was used as counterstaining for the nucleus. See related article, page 1433.

Circulation Research
Pages: e152 - e153
PubMed: 23625949

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Published online: 26 April 2013
Published in print: 24 May 2013

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Lisa J. Martin
Department of Medicine
Edward Lau
Departments of Medicine and Physiology
Harpreet Singh
Department of Anesthesiology
Laurent Vergnes
Department of Human Genetics
Lawrence Castellani
Department of Medicine
Peipei Ping
Departments of Medicine and Physiology
Karen Reue
Department of Human GeneticsMolecular Biology Institute
Enrico Stefani
Department of Anesthesiology
Thomas A. Drake
Department of Pathology and Laboratory Medicine
Kristina Bostrom
Department of Medicine
Aldons J. Lusis
Departments of Medicine, Human Genetics, and Microbiology, Immunology and Molecular GeneticsDavid Geffen School of Medicine University of CaliforniaLos Angeles, CA

Notes

Letters to the Editor will be published, if suitable, as space permits. They should not exceed 1000 words (typed double-spaced) in length and may be subject to editing or abridgment.

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Sources of Funding

This work was supported by funding from the NIH (HL30568, S10RR026744, HL088640, and HHSN268201000035C) and American Heart Association (11SDG7230059, 11POST7300060, and 12PRE11610024).

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  1. PXE, a Mysterious Inborn Error Clarified, Trends in Biochemical Sciences, 44, 2, (125-140), (2019).https://doi.org/10.1016/j.tibs.2018.10.005
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  2. Is ABCC6 a genuine mitochondrial protein?, BMC Research Notes, 6, 1, (2013).https://doi.org/10.1186/1756-0500-6-427
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