Metaxin 1 interacts with metaxin 2, a novel related protein associated with the mammalian mitochondrial outer membrane

Abstract

A recently described protein, metaxin 1, serves as a component of a preprotein import complex in the outer membrane of the mammalian mitochondrion. A yeast two-hybrid screen with metaxin 1 as bait has now identified a novel protein, which we have termed metaxin 2, as a metaxin 1-binding protein. Metaxin 2 shares 29% identity with metaxin 1 at the amino acid level, but metaxin 2, unlike metaxin 1, lacks a C-terminal mitochondrial outer membrane signal-anchor domain. Two C. elegans hypothetical proteins, CelZC97.1 and CelF39B2.i, share high sequence similarity with metaxin 2 and metaxin 1, respectively, and likely represent the C. elegans orthologs. Affinity-purified antibodies against metaxin 2 were prepared against the recombinant protein produced in E. coli and were used to analyze the subcellular distribution of metaxin 2. In subcellular fractions of mouse liver, a 29 kD immunoreactive protein, consistent in size with the predicted translation product of metaxin 2 cDNA, was found solely in mitochondria. Alkali extraction of mitochondria indicated that metaxin 2 is peripherally associated with mitochondrial membranes. Metaxin 2 in intact mitochondria was susceptible to digestion with proteinase K, indicating that metaxin 2 is located on the cytosolic face of the mitochondrial outer membrane. Finally, baculoviruses encoding a His6-tagged metaxin 2 and an untagged metaxin 1 lacking its C-terminal transmembrane domain were produced and used separately or in combination to infect Sf21 insect cells. Metaxin 1 bound to a Ni2+-chelate affinity column only in the presence of metaxin 2, indicating that metaxin 1 and metaxin 2 interact when overexpressed in insect cells. These results suggest that metaxin 2 is bound to the cytosolic face of the mitochondrial outer membrane by means of its interaction with membrane-bound metaxin 1, and that this complex may play a role in protein import into mammalian mitochondria.

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

“…Animals possess METAXIN1 and METAXIN2, which display just 36 and 28% sequence similarity to yeast SAM37 and SAM35, respectively (Mus musculus sequences). METAXIN2 was demonstrated to interact with METAXIN1 and to be peripherally associated with the mitochondrial outer membrane (Armstrong et al, 1999), akin to yeast SAM35 (Waizenegger et al, 2004). Taken together, the sequence similarity and protein-protein interactions suggest that the animal METAXIN proteins are orthologs of SAM35 and SAM37.…”

The Armadillo Repeat-containing Protein, ARMCX3, Physically and Functionally Interacts with the Developmental Regulatory Factor Sox10

“…Pellets from four aliquots of the mitochondrial preparations were resuspended in 2 mM HEPES, pH 7.4, 220 mM mannitol, 70 mM sucrose and then digested with varying concentrations of proteinase K for 90 min at room temperature. After proteinase K digestion, mitochondria were pelleted and washed as previously described (28). Western blot analysis was done to detect the distribution of Sox10, ARMCX3, and COX IV (Fig.…”

TOM22, a core component of the mitochondria outer membrane protein translocation pore, is a mitochondrial receptor for the proapoptotic protein Bax

“…Antibodies raised against metaxin 1 and TOM20 were obtained as described, respectively, by Saeki et al, 24 Armstrong et al 36 and Armstrong and Bornstein. 37 The mitochondrion-selective probe MitoTracker Green-FM was obtained from Molecular Probes (Interchim, Montlucon, France), and fluorogenic peptide Ac-DEVD-AMC was from Bachem (Voisins, France). Biochemical reagents used in this study were obtained from Sigma.…”