Functional Analysis of Human Metaxin in Mitochondrial Protein Import in Cultured Cells and Its Relationship with the Tom Complex

Abdul, Khaleque Md.; Terada, Kazutoyo; Yano, Masato; Ryan, Michael; Streimann, Illo Christian Arnold.; Hoogenraad, Nicholas J.; Mori, Masataka

doi:10.1006/bbrc.2000.3589

Cited by 41 publications

(28 citation statements)

References 32 publications

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“…2A, lower panels). To determine whether in vitro imported 35 S-Tom7 had assembled into a complex, we used Blue Native-PAGE (BN-PAGE) of digitonin-lysed mitochondria, which allows separation and visualization of mitochondrial translocase complexes (15,20,43,44). The radiolabeled Tom7 precursor assembled into an ϳ380-kDa complex independent of a ⌬ (Fig.…”

Section: Resultsmentioning

confidence: 99%

Insertion and Assembly of Human Tom7 into the Preprotein Translocase Complex of the Outer Mitochondrial Membrane

Johnston¹,

Hoogenraad²,

Dougan³

et al. 2002

Journal of Biological Chemistry

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128

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Tom7 is a component of the translocase of the outer mitochondrial membrane (TOM) and assembles into a general import pore complex that translocates preproteins into mitochondria. We have identified the human Tom7 homolog and characterized its import and assembly into the mammalian TOM complex. Tom7 is imported into mitochondria in a nucleotide-independent manner and is anchored to the outer membrane with its C terminus facing the intermembrane space. Unlike studies in fungi, we found that human Tom7 assembles into an ϳ120-kDa import intermediate in HeLa cell mitochondria. To detect subunits within this complex, we employed a novel supershift analysis whereby mitochondria containing newly imported Tom7 were incubated with antibodies specific for individual TOM components prior to separation by blue native electrophoresis. We found that the 120-kDa complex contains Tom40 and lacks receptor components. This intermediate can be chased to the stable ϳ380-kDa mammalian TOM complex that additionally contains Tom22. Overexpression of Tom22 in HeLa cells results in the rapid assembly of Tom7 into the 380-kDa complex indicating that Tom22 is rate-limiting for TOM complex formation. These results indicate that the levels of Tom22 within mitochondria dictate the assembly of TOM complexes and hence may regulate its biogenesis.

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Section: Resultsmentioning

confidence: 99%

Insertion and Assembly of Human Tom7 into the Preprotein Translocase Complex of the Outer Mitochondrial Membrane

Johnston¹,

Hoogenraad²,

Dougan³

et al. 2002

Journal of Biological Chemistry

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128

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“…Human mitochondria contain the proteins metaxin 1 and 2 that share limited homology to Sam37 and -35, respectively (25,53,54), although metaxin 1 does not form a discrete complex on BN-PAGE (55). A human Sam50 homologue has been identified at the level of protein sequence (21), but not characterized at the functional level.…”

Section: Human Tom40mentioning

confidence: 99%

Dissection of the Mitochondrial Import and Assembly Pathway for Human Tom40

Humphries

Streimann

Stojanovski

et al. 2005

Journal of Biological Chemistry

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173

142

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Tom40 is the channel-forming subunit of the translocase of the mitochondrial outer membrane (TOM complex), essential for protein import into mitochondria. Tom40 is synthesized in the cytosol and contains information for its mitochondrial targeting and assembly. A number of stable import intermediates have been identified for Tom40 precursors in fungi, the first being an association with the sorting and assembly machinery (SAM) of the outer membrane. By examining the import pathway of human Tom40, we have been able to elucidate additional features in its import. We identify that Hsp90 is involved in delivery of the Tom40 precursor to mitochondria in an ATP-dependent manner. The precursor then forms its first stable intermediate with the outer face of the TOM complex before its membrane integration and assembly. Deletion of an evolutionary conserved region within Tom40 disrupts the TOM complex intermediate and causes it to stall at a new complex in the intermembrane space that we identify to be the mammalian SAM. Unlike its fungal counterparts, the human Tom40 precursor is not found stably arrested at a SAM intermediate. Nevertheless, we show that Tom40 assembly is reduced in mitochondria depleted of human Sam50. These findings are discussed in context with current models from fungal studies.

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“…Plants also display differences in the SAM complex in comparison to yeast; in plants, only SAM50 can be clearly identified by sequence similarity (Lister et al, 2005). Arabidopsis METAXIN was identified by sequence similarity to the human METAXIN 1 protein; the latter displays limited sequence similarity to yeast SAM37 and has been implicated in mitochondrial protein import (Armstrong et al, 1997;Abdul et al, 2000). METAXIN1 and METAXIN2 in mammals have been reported to be involved in the import of b-barrel proteins but in a different complex compared with SAM50 (Kozjak-Pavlovic et al, 2007).…”

Section: Introductionmentioning

confidence: 99%

Functional Definition of Outer Membrane Proteins Involved in Preprotein Import into Mitochondria

et al. 2007

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The role of plant mitochondrial outer membrane proteins in the process of preprotein import was investigated, as some of the principal components characterized in yeast have been shown to be absent or evolutionarily distinct in plants. Three outer membrane proteins of Arabidopsis thaliana mitochondria were studied: TOM20 (translocase of the outer mitochondrial membrane), METAXIN, and mtOM64 (outer mitochondrial membrane protein of 64 kD). A single functional Arabidopsis TOM20 gene is sufficient to produce a normal multisubunit translocase of the outer membrane complex. Simultaneous inactivation of two of the three TOM20 genes changed the rate of import for some precursor proteins, revealing limited isoform subfunctionalization. Inactivation of all three TOM20 genes resulted in severely reduced rates of import for some but not all precursor proteins. The outer membrane protein METAXIN was characterized to play a role in the import of mitochondrial precursor proteins and likely plays a role in the assembly of β-barrel proteins into the outer membrane. An outer mitochondrial membrane protein of 64 kD (mtOM64) with high sequence similarity to a chloroplast import receptor was shown to interact with a variety of precursor proteins. All three proteins have domains exposed to the cytosol and interacted with a variety of precursor proteins, as determined by pull-down and yeast two-hybrid interaction assays. Furthermore, inactivation of one resulted in protein abundance changes in the others, suggesting functional redundancy. Thus, it is proposed that all three components directly interact with precursor proteins to participate in early stages of mitochondrial protein import.

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Functional Analysis of Human Metaxin in Mitochondrial Protein Import in Cultured Cells and Its Relationship with the Tom Complex

Cited by 41 publications

References 32 publications

Insertion and Assembly of Human Tom7 into the Preprotein Translocase Complex of the Outer Mitochondrial Membrane

Insertion and Assembly of Human Tom7 into the Preprotein Translocase Complex of the Outer Mitochondrial Membrane

Dissection of the Mitochondrial Import and Assembly Pathway for Human Tom40

Functional Definition of Outer Membrane Proteins Involved in Preprotein Import into Mitochondria

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