Abstract
ATP binding cassette (ABC) transporters are a diverse superfamily of energy-dependent membrane translocases. Although responsible for the majority of transmembrane transport in bacteria, they are relatively uncommon in eukaryotic mitochondria. Organellar trafficking and import, in addition to quaternary structure assembly, of mitochondrial ABC transporters is poorly understood and may offer explanations for the paucity of their diversity. Here we examine these processes in ABCB10 (ABC-me), a mitochondrial inner membrane erythroid transporter involved in heme biosynthesis. We report that ABCB10 possesses an unusually long 105-amino acid mitochondrial targeting presequence (mTP). The central subdomain of the mTP (amino acids (aa) 36-70) is sufficient for mitochondrial import of enhanced green fluorescent protein. The N-terminal subdomain (aa 1-35) of the mTP, although not necessary for the trafficking of ABCB10 to mitochondria, participates in the proper import of the molecule into the inner membrane. We performed a series of amino acid mutations aimed at changing specific properties of the mTP. The mTP requires neither arginine residues nor predictable alpha-helices for efficient mitochondrial targeting. Disruption of its hydrophobic character by the mutation L46Q/I47Q, however, greatly diminishes its efficacy. This mutation can be rescued by cryptic downstream (aa 106-715) mitochondrial targeting signals, highlighting the redundancy of this protein's targeting qualities. Mass spectrometry analysis of chemically cross-linked, immunoprecipitated ABCB10 indicates that ABCB10 embedded in the mitochondrial inner membrane homodimerizes and homo-oligomerizes. A deletion mutant of ABCB10 that lacks its mTP efficiently targets to the endoplasmic reticulum. Quaternary structure assembly of ABCB10 in the ER appears to be similar to that in the mitochondria.
Highlights
ABC1 transporters comprise a large and diverse family of membrane translocases [1]
N-terminal sequencing performed on the band identified the first 17 amino acids as LGNDSQRRPAATGRSEV, which correspond to ABCB10 aa 106 –123 (Fig. 1B)
The mitochondrial targeting presequence (mTP) of ABCB10 Is Necessary for Mitochondrial Import of ABCB10 and Sufficient for Import of EGFP—To explore the mitochondrial targeting and import properties of ABCB10 and its mTP, EGFP was fused at their C termini, and the proteins were expressed in HEK 293T cells
Summary
Cell Culture and Transfection—HEK 293T and COS cells were cultured in low glucose DMEM without phenol red (Invitrogen) supplemented with 10% standard fetal bovine serum (Hyclone, Logan, UT), 2 mM L-glutamine (Invitrogen), and antibiotics at 37 °C in 5% CO2, 95% air atmosphere. It was performed on all constructs that targeted partially or entirely to mitochondria Those constructs that targeted to mitochondria but were not fully imported are mentioned under “Results.” To accomplish extramitochondrial protein digestion, gradient-purified mitochondria were incubated with 100 g/ml proteinase K (Sigma) on ice for 30 min. Mitochondria were resuspended in buffer C (0.6 M sorbitol (Fisher), 20 mM HEPES, pH 7.4) supplemented with 2ϫ CompleteTM protease inhibitor mixture (Roche Applied Science), 2 mM phenylmethylsulfonyl fluoride, and 5.5% (v/v) trichloroacetic acid (Sigma), heated to 70 °C for 5 min, and incubated on ice for an additional 5 min. Tioned under “Results.” For whole cell protein analysis, cells were lysed in buffer C, supplemented with 0.1% (v/v) SDS and 2 M pepstatin A (Roche Applied Science) to prevent protein degradation. Protein was precipitated using 5.5% (v/v) trichloroacetic acid and immediately separated by SDS-PAGE
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