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Functional analysis of the conserved mitochondrial protein Mco76
Functional analysis of the conserved mitochondrial protein Mco76
Mitochondria execute a multitude of functions in the cell. To maintain their form and function, they need to form a complex membrane system composed of the mitochondrial outer membrane and the mitochondrial inner membrane. Contact sites between mitochondria and other organelles or between the inner mitochondrial membrane (IM) and the outer mitochondrial membrane (OM) are crucial for biogenesis and maintaining the mitochondrial membrane systems. These contacts sites are protein complexes that facilitate the transport of phospholipids and metabolites from other organelles to mitochondria and between mitochondrial membranes. Phospholipids reached to the mitochondria will be further directed to the proper submitochondrial localization, where they will be used for downstream processes. Mco76 is a member of the UbiB protein family containing an ACDK-like kinase (ATPase) domain. Interestingly, Mco76 is conserved throughout the evolution from yeast to human. The study presented here describes a functional analysis of Mco76 by studying its genetic interactions and biochemical interactions. In addition, it provides new insights into the function of Mco76 in the transfer of lipids between the IM and the OM. In this study, evidence was provided that Mco76 is present in the IM of mitochondria while its C-terminus is located in the mitochondrial intermembrane space (IMS). An immunoprecipitation assay was deployed to investigate biochemical interactions of Mco76. For the first time, Por1, Om14, and Om45 from the OM were identified as the protein interactors of Mco76. Hence, it was concluded that a novel contact site between the mitochondrial inner and outer membranes was discovered, providing potential hints into the function of Mco76. The genetic interactions of MCO76 were investigated to understand the role of Mco76 and the contact site formed by its interactions. Deleting of MCO76 from cells in which the CL pathway is impaired leads to a growth phenotype indicating MCO76 plays a role in phospholipid homoeostasis. Although available literature suggests that Mco76 is involved in the import of CoQ from mitochondria, genetic interactions of MCO76 suggest that MCO76 and the contact site formed by its interactions have broader functions in the cell. This idea was further supported by showing that the deletion of MCO76 reduces phosphatidic acid (PA) in mitochondria. In addition, it was shown that overexpression of MCO76 impairs mitochondrial architecture and alters mitochondrial morphology. Therefore, it was concluded that the defined levels of Mco76 are essential for mitochondrial architecture and morphology. Furthermore, it was shown that deletion of MCP2 rescues the growth defect of cells lacking Mco76 and Ups1. This further indicates that an equilibrium between the Mco76 and Mcp2 is necessary when the mitochondrial phospholipid composition is disturbed. This idea is consistent with the model proposed by Kemmerer that Mcp2 and Mco76 function reciprocally in importing and exporting CoQ from mitochondria. Overall, the study here established that Mco76 forms a contact site between the mitochondrial inner and outer membranes by interacting with Por1, om14, and Por1. Based on these findings, a model was proposed in which Mco76 facilitates the transfer of CoQ and phospholipids between the IM and the OM.
mitochondria, phospholipid homeostasis, contact site, mitochondria architecture, Porin proteins, Cardiolipin
Khosravi, Siavash
2022
English
Universitätsbibliothek der Ludwig-Maximilians-Universität München
Khosravi, Siavash (2022): Functional analysis of the conserved mitochondrial protein Mco76. Dissertation, LMU München: Faculty of Biology
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Abstract

Mitochondria execute a multitude of functions in the cell. To maintain their form and function, they need to form a complex membrane system composed of the mitochondrial outer membrane and the mitochondrial inner membrane. Contact sites between mitochondria and other organelles or between the inner mitochondrial membrane (IM) and the outer mitochondrial membrane (OM) are crucial for biogenesis and maintaining the mitochondrial membrane systems. These contacts sites are protein complexes that facilitate the transport of phospholipids and metabolites from other organelles to mitochondria and between mitochondrial membranes. Phospholipids reached to the mitochondria will be further directed to the proper submitochondrial localization, where they will be used for downstream processes. Mco76 is a member of the UbiB protein family containing an ACDK-like kinase (ATPase) domain. Interestingly, Mco76 is conserved throughout the evolution from yeast to human. The study presented here describes a functional analysis of Mco76 by studying its genetic interactions and biochemical interactions. In addition, it provides new insights into the function of Mco76 in the transfer of lipids between the IM and the OM. In this study, evidence was provided that Mco76 is present in the IM of mitochondria while its C-terminus is located in the mitochondrial intermembrane space (IMS). An immunoprecipitation assay was deployed to investigate biochemical interactions of Mco76. For the first time, Por1, Om14, and Om45 from the OM were identified as the protein interactors of Mco76. Hence, it was concluded that a novel contact site between the mitochondrial inner and outer membranes was discovered, providing potential hints into the function of Mco76. The genetic interactions of MCO76 were investigated to understand the role of Mco76 and the contact site formed by its interactions. Deleting of MCO76 from cells in which the CL pathway is impaired leads to a growth phenotype indicating MCO76 plays a role in phospholipid homoeostasis. Although available literature suggests that Mco76 is involved in the import of CoQ from mitochondria, genetic interactions of MCO76 suggest that MCO76 and the contact site formed by its interactions have broader functions in the cell. This idea was further supported by showing that the deletion of MCO76 reduces phosphatidic acid (PA) in mitochondria. In addition, it was shown that overexpression of MCO76 impairs mitochondrial architecture and alters mitochondrial morphology. Therefore, it was concluded that the defined levels of Mco76 are essential for mitochondrial architecture and morphology. Furthermore, it was shown that deletion of MCP2 rescues the growth defect of cells lacking Mco76 and Ups1. This further indicates that an equilibrium between the Mco76 and Mcp2 is necessary when the mitochondrial phospholipid composition is disturbed. This idea is consistent with the model proposed by Kemmerer that Mcp2 and Mco76 function reciprocally in importing and exporting CoQ from mitochondria. Overall, the study here established that Mco76 forms a contact site between the mitochondrial inner and outer membranes by interacting with Por1, om14, and Por1. Based on these findings, a model was proposed in which Mco76 facilitates the transfer of CoQ and phospholipids between the IM and the OM.