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MIC26 and MIC27 are bona fide subunits of the MICOS complex in mitochondria and do not exist as glycosylated apolipoproteins

Impairments of mitochondrial functions are linked to human ageing and pathologies such as cancer, cardiomyopathy, neurodegeneration and diabetes. Specifically, aberrations in ultrastructure of mitochondrial inner membrane (IM) and factors regulating them are linked to diabetes. The development of di...

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Autores principales: Lubeck, Melissa, Derkum, Nick H., Naha, Ritam, Strohm, Rebecca, Driessen, Marc D., Belgardt, Bengt-Frederik, Roden, Michael, Stühler, Kai, Anand, Ruchika, Reichert, Andreas S., Kondadi, Arun Kumar
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10243636/
https://www.ncbi.nlm.nih.gov/pubmed/37279200
http://dx.doi.org/10.1371/journal.pone.0286756
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author Lubeck, Melissa
Derkum, Nick H.
Naha, Ritam
Strohm, Rebecca
Driessen, Marc D.
Belgardt, Bengt-Frederik
Roden, Michael
Stühler, Kai
Anand, Ruchika
Reichert, Andreas S.
Kondadi, Arun Kumar
author_facet Lubeck, Melissa
Derkum, Nick H.
Naha, Ritam
Strohm, Rebecca
Driessen, Marc D.
Belgardt, Bengt-Frederik
Roden, Michael
Stühler, Kai
Anand, Ruchika
Reichert, Andreas S.
Kondadi, Arun Kumar
author_sort Lubeck, Melissa
collection PubMed
description Impairments of mitochondrial functions are linked to human ageing and pathologies such as cancer, cardiomyopathy, neurodegeneration and diabetes. Specifically, aberrations in ultrastructure of mitochondrial inner membrane (IM) and factors regulating them are linked to diabetes. The development of diabetes is connected to the ‘Mitochondrial Contact Site and Cristae Organising System’ (MICOS) complex which is a large membrane protein complex defining the IM architecture. MIC26 and MIC27 are homologous apolipoproteins of the MICOS complex. MIC26 has been reported as a 22 kDa mitochondrial and a 55 kDa glycosylated and secreted protein. The molecular and functional relationship between these MIC26 isoforms has not been investigated. In order to understand their molecular roles, we depleted MIC26 using siRNA and further generated MIC26 and MIC27 knockouts (KOs) in four different human cell lines. In these KOs, we used four anti-MIC26 antibodies and consistently detected the loss of mitochondrial MIC26 (22 kDa) and MIC27 (30 kDa) but not the loss of intracellular or secreted 55 kDa protein. Thus, the protein assigned earlier as 55 kDa MIC26 is nonspecific. We further excluded the presence of a glycosylated, high-molecular weight MIC27 protein. Next, we probed GFP- and myc-tagged variants of MIC26 with antibodies against GFP and myc respectively. Again, only the mitochondrial versions of these tagged proteins were detected but not the corresponding high-molecular weight MIC26, suggesting that MIC26 is indeed not post-translationally modified. Mutagenesis of predicted glycosylation sites in MIC26 also did not affect the detection of the 55 kDa protein band. Mass spectrometry of a band excised from an SDS gel around 55 kDa could not confirm the presence of any peptides derived from MIC26. Taken together, we conclude that both MIC26 and MIC27 are exclusively localized in mitochondria and that the observed phenotypes reported previously are exclusively due to their mitochondrial function.
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spelling pubmed-102436362023-06-07 MIC26 and MIC27 are bona fide subunits of the MICOS complex in mitochondria and do not exist as glycosylated apolipoproteins Lubeck, Melissa Derkum, Nick H. Naha, Ritam Strohm, Rebecca Driessen, Marc D. Belgardt, Bengt-Frederik Roden, Michael Stühler, Kai Anand, Ruchika Reichert, Andreas S. Kondadi, Arun Kumar PLoS One Research Article Impairments of mitochondrial functions are linked to human ageing and pathologies such as cancer, cardiomyopathy, neurodegeneration and diabetes. Specifically, aberrations in ultrastructure of mitochondrial inner membrane (IM) and factors regulating them are linked to diabetes. The development of diabetes is connected to the ‘Mitochondrial Contact Site and Cristae Organising System’ (MICOS) complex which is a large membrane protein complex defining the IM architecture. MIC26 and MIC27 are homologous apolipoproteins of the MICOS complex. MIC26 has been reported as a 22 kDa mitochondrial and a 55 kDa glycosylated and secreted protein. The molecular and functional relationship between these MIC26 isoforms has not been investigated. In order to understand their molecular roles, we depleted MIC26 using siRNA and further generated MIC26 and MIC27 knockouts (KOs) in four different human cell lines. In these KOs, we used four anti-MIC26 antibodies and consistently detected the loss of mitochondrial MIC26 (22 kDa) and MIC27 (30 kDa) but not the loss of intracellular or secreted 55 kDa protein. Thus, the protein assigned earlier as 55 kDa MIC26 is nonspecific. We further excluded the presence of a glycosylated, high-molecular weight MIC27 protein. Next, we probed GFP- and myc-tagged variants of MIC26 with antibodies against GFP and myc respectively. Again, only the mitochondrial versions of these tagged proteins were detected but not the corresponding high-molecular weight MIC26, suggesting that MIC26 is indeed not post-translationally modified. Mutagenesis of predicted glycosylation sites in MIC26 also did not affect the detection of the 55 kDa protein band. Mass spectrometry of a band excised from an SDS gel around 55 kDa could not confirm the presence of any peptides derived from MIC26. Taken together, we conclude that both MIC26 and MIC27 are exclusively localized in mitochondria and that the observed phenotypes reported previously are exclusively due to their mitochondrial function. Public Library of Science 2023-06-06 /pmc/articles/PMC10243636/ /pubmed/37279200 http://dx.doi.org/10.1371/journal.pone.0286756 Text en © 2023 Lubeck et al https://creativecommons.org/licenses/by/4.0/This is an open access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
spellingShingle Research Article
Lubeck, Melissa
Derkum, Nick H.
Naha, Ritam
Strohm, Rebecca
Driessen, Marc D.
Belgardt, Bengt-Frederik
Roden, Michael
Stühler, Kai
Anand, Ruchika
Reichert, Andreas S.
Kondadi, Arun Kumar
MIC26 and MIC27 are bona fide subunits of the MICOS complex in mitochondria and do not exist as glycosylated apolipoproteins
title MIC26 and MIC27 are bona fide subunits of the MICOS complex in mitochondria and do not exist as glycosylated apolipoproteins
title_full MIC26 and MIC27 are bona fide subunits of the MICOS complex in mitochondria and do not exist as glycosylated apolipoproteins
title_fullStr MIC26 and MIC27 are bona fide subunits of the MICOS complex in mitochondria and do not exist as glycosylated apolipoproteins
title_full_unstemmed MIC26 and MIC27 are bona fide subunits of the MICOS complex in mitochondria and do not exist as glycosylated apolipoproteins
title_short MIC26 and MIC27 are bona fide subunits of the MICOS complex in mitochondria and do not exist as glycosylated apolipoproteins
title_sort mic26 and mic27 are bona fide subunits of the micos complex in mitochondria and do not exist as glycosylated apolipoproteins
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10243636/
https://www.ncbi.nlm.nih.gov/pubmed/37279200
http://dx.doi.org/10.1371/journal.pone.0286756
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