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Functional analysis of human metaxin in mitochondrial protein import in cultured cells and its relationship with the
1Department of Molecular Genetics, Kumamoto University School of Medicine, Honjo 2-2-1, Kumamoto, 860-0811, Japan.
Abstract:
Metaxin is an outer membrane protein of mammalian mitochondria which is suggested to be involved in protein import into the organelle. RNA blot analysis showed that distribution of metaxin mRNA in human tissues differs from that of mRNA for the translocase component Tom20. Effect of overexpression of human metaxin on mitochondrial preprotein import and processing in COS-7 cells was studied. Overexpression of metaxin resulted in impaired mitochondrial import of natural and chimeric preproteins and in their accumulation. We previously reported that overexpression of Tom20 in cultured cells causes inhibition of import of mitochondrial preprotein. Coexpression of metaxin with Tom20 had no further effect on the preprotein import. Overexpression of the cytosolic domain of metaxin also caused inhibition of preprotein import, although less strongly than the full-length metaxin. In blue native PAGE, Tom40, Tom22, and a portion of Tom20 migrated as a complex of approximately 400 kDa, and the other portion of Tom20 migrated in smaller forms of approximately 100 and approximately 40 kDa. On the other hand, metaxin migrated at a position of approximately 50 kDa. These results confirm earlier in vitro results that metaxin participates in preprotein import into mammalian mitochondria, and indicates that it does not associate with the Tom complex.
Insights
Metaxin, an outer mitochondrial protein, impairs preprotein import when overexpressed. This study confirms metaxin
Area of Science:
- Mitochondrial biology
- Protein import mechanisms
- Cellular and molecular biology
Background:
- Metaxin is an outer mitochondrial membrane protein implicated in organelle protein import.
- The distribution of metaxin mRNA in human tissues differs from that of the translocase component Tom20.
- Previous studies indicated that overexpression of Tom20 inhibits mitochondrial preprotein import.
Purpose of the Study:
- To investigate the role of human metaxin in mitochondrial protein import.
- To determine the effect of metaxin overexpression on preprotein import and processing in COS-7 cells.
- To examine the potential association of metaxin with the TOM complex.
Main Methods:
- Overexpression of human metaxin and its cytosolic domain in COS-7 cells.
- Analysis of mitochondrial preprotein import and processing.
- Blue native polyacrylamide gel electrophoresis (PAGE) to analyze protein complex formation.
Main Results:
- Overexpression of metaxin led to impaired mitochondrial import and accumulation of preproteins.
- Coexpression of metaxin with Tom20 did not result in further inhibition of preprotein import.
- Blue native PAGE showed metaxin migrating independently of the TOM complex (Tom40, Tom22, Tom20).
Conclusions:
- Metaxin plays a role in preprotein import into mammalian mitochondria.
- Metaxin does not appear to associate with the TOM complex.
- The cytosolic domain of metaxin also contributes to the inhibition of preprotein import.