Alternative splicing gives rise to different isoforms of the Neurospora crassa Tob55 protein that vary in their ability to insert beta-barrel proteins into the outer mitochondrial membrane

Abstract

Tob55 is the major component of the TOB complex, which is found in the outer membrane of mitochondria. A sheltered knockout of the tob55 gene was developed in Neurospora crassa. When grown under conditions that reduce the levels of the Tob55 protein, the strain exhibited a reduced growth rate and mitochondria isolated from these cells were deficient in their ability to import beta-barrel proteins. Surprisingly, Western blots of wild-type mitochondrial proteins revealed two bands for Tob55 that differed by approximately 4 kDa in their apparent molecular masses. Sequence analysis of cDNAs revealed that the tob55 mRNA is alternatively spliced and encodes three isoforms of the protein, which are predicted to contain 521, 516, or 483 amino acid residues. Mass spectrometry of proteins isolated from purified outer membrane vesicles confirmed the existence of each isoform in mitochondria. Strains that expressed each isoform of the protein individually were constructed. When cells expressing only the longest form of the protein were grown at elevated temperature, their growth rate was reduced and mitochondria isolated from these cells were deficient in their ability to assembly beta-barrel proteins.

Figure 1.—

Sheltered disruption of tob55. (A) Split marker transformants of heterokaryon HP1 were isolated and purified as described in the text. Serial dilutions of conidia produced by the transformants were tested on the indicated media to determine which nucleus carried the tob55 disruption. (B) Genotype of the sheltered heterokaryons Tob55KO-1 and Tob55KO-3. The box symbolizes a heterokaryotic cell with circles representing its component nuclei. Genetic markers important for maintenance, selection, or manipulation of the strain are shown (tob55, topogenesis of outer membrane β-barrel proteins; his, histidine; pan, pantothenate; mtr, methyltryptophan resistance; Bml, benomyl resistance). A mutation in the mtr gene results in resistance to fpa. Certain mutations in the Bml gene result in resistance to benomyl. The nucleus with histidine auxotrophy and fpa resistance (mtr) carries the tob55 deletion.

Figure 2.—

N. crassa Tob55 is involved in biogenesis of β-barrel proteins. (A) Mitochondria were isolated from the control strain (HP1) and the tob55 knockout sheltered heterokaryon strain (Tob55KO-3) following growth in the absence (−) or presence (+) of 400 μm fpa and histidine. Mitochondrial proteins (20 μg) were separated by SDS–PAGE, blotted to nitrocellulose, and analyzed by immunodecoration with antisera against the indicated proteins. Tob55 is present as two bands of 62 and 58 kDa, respectively. (B) As in A except mitochondria were isolated from the control (HP1) and two strains (T55his6-1, T55his6-3) obtained by rescue of the Δtob55, histidine-requiring nucleus from strain Tob55KO-3 by a genomic tob55 gene encoding an N-terminal hexahistidinyl tag. Blots were immunodecorated with Tob55 antiserum or penta-His antiserum. his, histidine.

Figure 3.—

Tob55-deficient mitochondria are defective in import of outer membrane β-barrel proteins. (A–E) Mitochondria were isolated from strain HP1 (control) and the tob55 knockout strain Tob55KO-3 (Tob55↓) following growth in the presence of 250 μm fpa plus histidine. For import assays, mitochondria were incubated with lysates containing radiolabeled mitochondrial precursors at 15° for the indicated times. Following a post-import proteinase K treatment, mitochondria were reisolated and subjected to SDS–PAGE. The gels were transferred to nitrocellulose and exposed to X-ray film and then a PhosphorImager screen. One sample from each strain was treated with trypsin prior to import (“pre trp”) to demonstrate receptor-dependent import. “lys” represents 33% of the total radioactivity added to each reaction. (F) Western blot showing the level of Tob55, Tom40, and Tom70 in mitochondria isolated from cultures grown in the presence of 250 μm fpa plus histidine.

Figure 4.—

Assembly of Tom40 and porin in Tob55↓ mitochondria. Radiolabeled Tom40 (A) or porin (B) precursor were incubated for 20 min at the indicated temperature with mitochondria isolated from the control strain HP1 (Ct) or Tob55KO-3 (55↓), both grown in the presence of fpa (250 μm) and histidine. Mitochondria were washed with 80 mm KCl, reisolated, and lysed in blue gel sample buffer containing 1% digitonin. The samples were subjected to blue native gel electrophoresis, blotted to PVDF membrane, and analyzed by autoradiography. (C and D) Antibody supershift experiments. Import was performed as for A and B with Tom40 (C) and porin (D) precursor proteins using mitochondria isolated from a wild-type strain (NCN 251). Following import at 25°, mitochondria were lysed with 1% digitonin. Reactions were then incubated with buffer alone (“No Ab”), affinity-purified antibody to Tob55, or affinity-purified antibody to D. melanogaster ATM protein as a negative control for 2 hr at 4° and then subjected to BNGE. The gel was blotted to PVDF membrane and examined by autoradiography. Apparent sizes of the intermediates are shown on the left. The position of the supershifted products is indicated with an arrow on the right.

Figure 5.—

Intron/exon structure of the tob55 gene. (A) The positions of all possible exons (rectangular boxes) and introns (solid lines) of tob55 are indicated. The number of codons is given in parentheses below the number of each exon. Possible 5′ splice sites are shown above the line and possible 3′ splice sites are shown below. Potential splice sites are numbered in sequence. Exon 2a is not separated from exon 2 by an intron, but alternative splicing may remove exon 2a from the remainder of exon 2 in some cases. (B) The three forms of the Tob55 protein that arise from alternative splicing. Shading is coded to correspond to the different exons shown in A. The solid bars under each isoform indicate peptides predicted to be generated by tryptic digestion that would be unique for each isoform of the protein. (C) Mass spectrometry of Tob55 isoforms. The peptides predicted to be generated by tryptic digestion as indicated in B are shown. Six His residues occur after the initial Met residue on the short form because the analysis was done on a His-tagged version of the protein. Arrows under the peptide sequences indicate the splice point between exon 1 and exon 3 for the short form, between exon 2 and exon 3 for the intermediate (“interm”) form, and between exon 2a and exon 3 for the long form. The five residues that make up exon 2a are underlined in the long form. The sequence of each peptide is followed by its predicted (“P”) mass and the mass that was determined experimentally (“E”) by mass spectrometry. The mass of the predominant peak corresponding to the unique peptide for the short form shows that oxidation of the peptide, probably at the N-terminal Met residue, has occurred. The predicted mass includes this consideration. The predicted and experimentally determined masses for the short and long peptides differ by <2 Da. This is within the limits of accuracy for these large peptides whose masses were determined by linear MALDI–TOF. The tracings from the appropriate regions of the mass spectra for each peptide are shown below.

Figure 6.—

Strains expressing only the long isoform of Tob55 have growth defects. Constructs expressing cDNA versions of the three different Tob55 splicing variants from the endogenous tob55 promoter were used to rescue the Δtob55 nucleus from strain Tob55KO-3. This resulted in strains ST55-2, IT55-8, and LT55-2 expressing the short, intermediate (“interm”), and long isoforms of the protein, respectively. (A) Mitochondria (30 μg) isolated from these three strains and the control strain (76-26) following growth at 30° were examined on Western blots to demonstrate that only one form of the protein was expressed in each strain. Porin was used as the loading control. (B) Serial dilutions of conidia from the strains described in A were spotted onto solid sorbose-containing medium and grown at 30° or 37°. (C) As in B, but conidia were spotted on plates containing either 100 or 500 mm NaCl and were grown at 30°. (D) The short and long strains were grown at 37° in liquid cultures containing 250 ml of Vogel's medium. Each flask was inoculated with 10⁶ conidia/ml. At the times indicated, the cultures were harvested and the pad of mycelium was dried and then weighed to give the dry weight of the culture. (E) The control, short, and long strains were grown in liquid medium at 30° and 37° for 24 hr. Mitochondria were isolated and examined for the presence of various mitochondrial proteins (indicated on the right) by Western blot analysis. Each lane contained 30 μg of mitochondrial protein.

Figure 7.—

Assembly of β-barrel proteins in mitochondria isolated from 37° cultures. (A) Mitochondria were isolated from strain 76-26 (control) and ST55-2 following 16 hr growth at 37° (inoculum of 10⁶ conidia/ml) and from LT55-2 after 24 hr at 37° using twice the amount of inoculum. Mitochondria were incubated with radiolabeled precursors of Tom40 or porin for 20 min at the indicated temperature and analyzed by BNGE. (B) As in A, except that LT55-2 was also grown for 68 hr with an inoculum of 10⁶ conidia/ml. The size of assembly products is indicated on the left. (C) Western blots analysis of mitochondrial proteins from the strains indicated following growth at 37° for the indicated times.

Figure 8.—

Alignment of N-terminal region of Tob55. The position of the residues present in the N. crassa long isoform, but absent in the small isoform, are indicated by the thin line above the alignment. Residues absent in the intermediate isoform are shown by the thick line. The start of the POTRA domain (see discussion), as defined previously (Sanchez-Pulido et al. 2003), is indicated by a plus sign (+) above the alignment. Residues that are identical in six or more of the seven species are shown against a solid background. Residues identical in four or five species are shown against a shaded background. NcL, long isoform of N. crassa Tob55; Cg, C. globosum; Gz, G. zeae; An, A. nidulans; Sc, S. cerevisiae; At, A. thaliana; Hs, H. sapiens.