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. 1998 Jun;18(6):3173-81.
doi: 10.1128/MCB.18.6.3173.

Role of the negative charges in the cytosolic domain of TOM22 in the import of precursor proteins into mitochondria

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Role of the negative charges in the cytosolic domain of TOM22 in the import of precursor proteins into mitochondria

F E Nargang et al. Mol Cell Biol. 1998 Jun.

Abstract

TOM22 is an essential mitochondrial outer membrane protein required for the import of precursor proteins into the organelles. The amino-terminal 84 amino acids of TOM22 extend into the cytosol and include 19 negatively and 6 positively charged residues. This region of the protein is thought to interact with positively charged presequences on mitochondrial preproteins, presumably via electrostatic interactions. We constructed a series of mutant derivatives of TOM22 in which 2 to 15 of the negatively charged residues in the cytosolic domain were changed to their corresponding amido forms. The mutant constructs were transformed into a sheltered Neurospora crassa heterokaryon bearing a tom22::hygromycin R disruption in one nucleus. All constructs restored viability to the disruption-carrying nucleus and gave rise to homokaryotic strains containing mutant tom22 alleles. Isolated mitochondria from three representative mutant strains, including the mutant carrying 15 neutralized residues (strain 861), imported precursor proteins at efficiencies comparable to those for wild-type organelles. Precursor binding studies with mitochondrial outer membrane vesicles from several of the mutant strains, including strain 861, revealed only slight differences from binding to wild-type vesicles. Deletion mutants lacking portions of the negatively charged region of TOM22 can also restore viability to the disruption-containing nucleus, but mutants lacking the entire region cannot. Taken together, these data suggest that an abundance of negative charges in the cytosolic domain of TOM22 is not essential for the binding or import of mitochondrial precursor proteins; however, other features in the domain are required.

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Figures

FIG. 1
FIG. 1
Structure of the TOM22 protein and mutants derived by site-directed mutagenesis of acidic residues. (Top) Putative structure of N. crassa TOM22 showing the cytosolic domain, the membrane-spanning region, and the intermembrane space (IMS) domain of the protein. The positions of all acidic and basic residues in the cytosolic domain are indicated. (Bottom) Charge neutralization mutant strains used in this study. These were derived by site-directed mutagenesis of various numbers of acidic residues in the cytosolic domain. The numbers of negative and positive charges present in each mutant as well as the net charge in the cytosolic domain are indicated.
FIG. 2
FIG. 2
Scheme for isolation of tom22 mutant strains. Strain ND-113-1 (40) (see Materials and Methods) is the sheltered heterokaryon represented by the box at the top. Nucleus 1 contains a tom22 gene disrupted by a gene encoding hyrgromycin resistance (hygR). In addition, nucleus 1 carries auxotrophy for histidine and a resistance gene for p-fluorophenylalanine (fpaR). It is possible to select for rescue of the tom22 deficiency in nucleus 1 when that nucleus is transformed with both a bleomycin resistance gene and a mutant tom22 gene when that mutant gene gives rise to a functional protein. The presence of histidine in the selective medium frees nucleus 1 from its dependence on nucleus 2 for growth, while the presence of FPA forces transformants to be greatly enriched for nucleus 1. Purification and nutritional testing of the colonies derived from transformation with any of the mutant forms of tom22 described in the legend to Fig. 1 revealed that all bleomycin-resistant transformants were incapable of growth on medium lacking histidine. Thus, all of the transformants were homokaryotic for nucleus 1 and all of the mutant forms of tom22 were able to restore TOM22 function.
FIG. 3
FIG. 3
Growth of wild-type strain 76-26 and mutant strains. (A) Colonies formed on sorbose-containing medium after 48 h of growth at 30°C are shown. The previously described CCHL-deficient strain (the cyt-2-1 strain) is shown as an example of a slowly growing mutant. (B) Mycelial elongation of strains 76-26 and 861 and the cyt-2-1 strain as measured in race tubes (see Materials and Methods) at 22°C. (C) Same as panel B, but with growth at 37°C.
FIG. 4
FIG. 4
Immunostaining of mitochondrial proteins in wild-type (76-26) and tom22 mutant strains. Equal amounts of mitochondrial protein were loaded in each lane. Proteins were separated by SDS-PAGE, transferred to a polyvinylidene difluoride membrane, and decorated with antiserum to the indicated proteins. (A) Levels of TOM complex components in the wild type and all charge neutralization mutant strains generated in this study. Alterations in many of the TOM22 mutants result in altered electrophoretic mobilities relative to that of wild-type TOM22. (B) Levels of TOM40 and TOM22 in three different isolates obtained following transformation of strain ND-113-1 with the 861 mutant form of tom22.
FIG. 5
FIG. 5
Import of [35S]methionine-labeled precursor proteins into isolated mitochondria. Mitochondria isolated from strain 861 (A), strain 100 (B), and wild-type control strain 76-26 were either pretreated with trypsin (+) or mock treated (m). Import was conducted at 10°C for the time periods indicated. Following import reactions, mitochondria were reisolated and subjected to SDS-PAGE. The gels were blotted to a polyvinylidene difluoride membrane and subjected to autoradiography. The precursors used in the import reactions are indicated on the left. The leftmost lane for each precursor contained 33% of the input lysate used in each import reaction. cyt c1, cytochrome c1; p, m, and i, positions of the precursor, mature, and intermediate forms of the preproteins, respectively. (C) Same as for panel A, except with import performed at 25°C.
FIG. 6
FIG. 6
Charges in TOM22 are not required for preprotein binding at the cis site or for presequence translocation to the trans site of the outer membrane. OMV (5 μg of protein per sample) were isolated from the indicated tom22 mutant strains. Binding of pSu9-DHFR was performed in 100 μl of binding buffer (0.25-mg/ml bovine serum albumin, 2.5 mM MgCl2, 20 mM KCl, 10 mM morpholinepropanesulfonic acid [MOPS]-KOH [pH 7.2]) in the absence (trans site binding) or presence (cis site binding) of 1 mM NADPH and 1 μM methotrexate for 15 min at 25 or 0°C, respectively. Following incubation, samples were diluted with 700 μl of buffer (10 mM MOPS-KOH, 1 mM EDTA [pH 7.2]) containing 120 mM KCl (trans site binding) or 20 mM KCl (cis site binding). OMV were reisolated by centrifugation for 20 min at 125,000 × g, and the pellets were subjected to SDS-PAGE. Bound pSu9-DHFR was quantitated by PhosphorImager analysis. The amount of pSu9-DHFR bound to the wild-type strain (76-26) was set to 100%. Data shown are the averages of seven trials. Standard deviations of the different data sets ranged from 15 to 25%. White bars, cis site binding; black bars, trans site binding.
FIG. 7
FIG. 7
Alignment of N. crassa and S. cerevisiae TOM22 proteins. Only the region altered in mutant 95 and the corresponding yeast mutant (6) is shown. The mutations generated in each of the two species are indicated in parentheses. |, identical amino acids; :, chemically similar amino acids.
FIG. 8
FIG. 8
Import of preproteins into mitochondria isolated from strain 95 and wild-type strain 76-26. Details are as described in the legend to Fig. 5.
FIG. 9
FIG. 9
Growth of tom22 deletion strains. Mycelial elongation of the wild-type strain (76-26) and the Δ2-28 and Δ32-44 mutant strains was measured in race tubes at 22°C (A) and at 37°C (B). The curves for 76-26 and Δ32-44 in panel B are entirely overlapping.
FIG. 10
FIG. 10
Immunostaining of TOM complex proteins in mitochondria isolated from tom22 deletion strains. Details are as described in the legend to Fig. 4.

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