Table 1.
Strains used in this study.
Figure 1.
Isolation and characterization of N. crassa strains with reduced levels of Tob37 or Tob38.
(a) Sheltered heterokaryons with deletions of either tob37 or tob38 in one nucleus of the heterokaryon were constructed using a split marker approach. Boxes symbolize heterokaryons while circles within the boxes represent the different component nuclei of the heterokaryon. The Figure shows an example for tob37, but the process was identical for tob38. The starting heterokaryon (HP1) contained nuclei with different genetic markers: either his-3 and mtr (provides resistance to fpa) or pan-2 and Bml (provides resistance to benomyl). Strains chosen for further work carried the knockouts in the his-3 mtr nucleus (see Methods). (b) Serial dilutions of conidiaspores (actual numbers spotted shown at top of panel) from the strains indicated on the left were spotted onto plates containing either minimal medium (min), which maintains both nuclei of the heterokaryon approximately equally; minimal medium containing pantothenate and benomyl (pan ben), which forces the nucleus carrying benomyl resistance (Bml, Figure 1A) to predominate the culture; or minimal medium containing histidine plus fpa (his fpa), which forces the nucleus carrying fpa resistance (mtr, Figure 1A) to predominate the culture. The control was strain HP1. (c) Cells from the indicated strains (top of panel) were grown in the presence of histidine (His) and fpa to force the predominance of the nucleus bearing the deletion of either tob37 or tob38. This results in reduction of the levels of Tob37 or Tob38, respectively. Mitochondria were isolated and subjected to SDS-PAGE followed by transfer to nitrocellulose, and immunodecoration with the anitbodies indicated on the left. The control strain was HP1. Multiple bands in the Tob55 lane correspond to different isoforms of the protein [27]. (d) Mitochondria isolated from the indicated strains were either untreated (Mitos) or incubated in the presence of proteinase K (Mitos + pK) for 15 min. Mitochondrial proteins were then subjected to SDS-PAGE and western blotting. The blot was examined for the presence of Tom70 and the intermembrane space protein CCHL. (e) As in panel C, except strains were grown in minimal medium which maintains the numbers of both types of nuclei in the culture approximately equally. (f) Conidia produced from the sheltered heterokaryons (ΔTob37 and ΔTob38) were streaked onto medium containing histidine and pantothenate. Individual colonies were isolated and tested for nutritional requirements to determine if they were histidine-requiring homokaryons (His-req), pantothenate requiring homokaryons (Pan-req), or heterokaryons (Het).
Figure 2.
Import/assembly of mitochondrial precursor proteins into mitochondria deficient in Tob37 or Tob38.
(a) Radiolabeled matrix precursor F1β and inner membrane precursor AAC were incubated (for 5 min or 20 min, as indicated) with mitochondria isolated from heterokaryotic strains (indicated at the top of the panel) grown in the presence of histidine and fpa to reduce levels of Tob37 or Tob38 in the respective mutants. Following import, mitochondria were subjected to SDS-PAGE. Proteins were transferred to nitrocellulose membrane, and import was analyzed by autoradiography. (control, strain HP1; lys, 33% of the radiolabeled lysate added to each import reaction; bp, “bypass import” in mitochondria treated with trypsin to remove surface receptors prior to the import reaction; p, precursor protein; m, mature protein.) (b) Radiolabelled Tom40 precursor was incubated for 5 min and 20 min with mitochondria isolated from the strains indicated (top of panel) grown in the presence of histidine and fpa. Mitochondria were dissolved in 1% digitonin and subjected to BNGE. The proteins were transferred to PVDF membrane and analyzed by autoradiography. The size of the mature TOM complex (400 kDa), and assembly intermediates I (250 kDa) and II (100 kDa) are indicated on the left. * indicates an undefined band. (c) Tom40 was imported into mitochondria isolated from the strains indicated for 20 min. Following import, proteinase K was added to each import reaction for 15 min on ice. PMSF was added to inactivate the proteinase, each reaction was divided into equal halves, and mitochondria were pelleted. One half was suspended in SDS-PAGE cracking buffer (Mitos). The other half was suspended in sodium carbonate (pH 11.5) and incubated on ice for 30 min. The membrane sheets were pelleted and suspended in cracking buffer (Carb pellet). Both sets of reactions were subjected to SDS-PAGE and the proteins were transferred to nitrocellulose membrane and examined by autoradiography. The positions of Tom40 and the 26 kDa and 12 kDa fragments generated by proteinase K digestion are indicated. (d) As in panel B except that mitochondria were incubated with the radiolabeled precursor of porin. The numbers on the left indicate the position of molecular weight markers. (e) Assembly of Tom22. As in panel D, except mitochondria were incubated with radiolabeled Tom22.
Figure 3.
(a) Number and protein content of N. crassa TOB complexes. Mitochondria were isolated from strains expressing only His-tagged versions (instead of the endogenous versions) of either Tob55 (Tob55HT), Tob38 (Tob38HT), or Tob37 (Tob37HT) as indicated for each lane at the top of the panel. TOB complexes were purified using Ni-NTA resin. Purified complexes were subjected to BNGE, transferred to PVDF membrane, and decorated with antibodies to Tob55, Mdm10, Tob37, or Tob38 as indicated at the bottom of each panel. The position of molecular weight markers (kDa) is shown on the left and the estimated size (kDa) of complexes is shown on the right. (b) TOB complex was purified from a strain carrying His-tagged Tob55 and subjected to first dimension BNGE (1st dim) in two separate lanes of the gel. One lane was transferred to PVDF, and decorated with antibody to Tob55 (top lane in panel). The second lane was removed for second dimension (2nd dim) electrophoresis by SDS-PAGE as described in the Methods. Following SDS-PAGE, the gel was transferred to nitrocellulose. The membrane was cut into strips corresponding to the molecular weights of Tob55, Tob38, and Tob37 and probed with antibodies to those proteins, respectively (indicated on the left). Sizes of TOB complex following 1st dimension BNGE are indicated at the top of the panel. (c) As in panel B, except the purification was performed using mitochondria containing His-tagged Tob38 and the SDS-PAGE blot was examined with antibodies to Tob38 and Mdm10. (d) As in panel B except that whole mitochondria were examined for the presence of TOB complexes. (e) As in panel A, except TOB complex was purified from mitochondria isolated from cells expressing only His-tagged versions of different Tob55 isoforms [27]: short Tob55 (Tob55 Short HT), intermediate Tob55 (Tob55 Int HT), or long Tob55 (Tob55 Long HT) as indicated at the top of the panels. Blots were immunodecorated with the antibodies indicated at the bottom of the panels.
Figure 4.
(a) Mitochondria isolated from wild type cells (strain 76-26) were treated with proteinase K (+pK) as in Figure 2C or not (-pK). Mitochondria were washed and subjected to SDS-PAGE. The gel was blotted to nitrocellulose and examined by immunodecoration with the antibodies indicated on the left. (b) Mitochondria from the control strain (76-26) were subjected to extraction with 0.1 M sodium carbonate at the pHs indicated at the top of the panel. Following the treatment, membrane sheets were pelleted and supernatants were subjected to trichloroacetic acid precipitation. Membrane pellets (p) and supernatant precipitates (s) were subjected to SDS-PAGE. Proteins in the gel were transferred to nitrocellulose and the membrane was examined with the antibodies indicated on the left. (c) As in panel B except extractions were done at pH 11.0 from control (HP1), ΔTob37, and ΔTob38 strains grown in the presence of histidine and fpa. The three lanes on the left show the protein levels in whole mitochondria (mitos), while the six lanes on the right show the pellets and supernatants resulting from alkali extraction.
Figure 5.
Role of predicted TMDs of Tob37.
(a) The WT row shows the sequence of the 63 amino acids at the C-terminus of wild type N. crassa Tob37. ΔTMD1 is the deletion constructed for the first possible transmembrane domain and is the name of the strain expressing this form of Tob37. Similarly for CHD, the second possible TMD found at the C-terminus of the protein, and for ΔT+C, the deletion of the last 56 amino acids of the protein which removes both possible TMDs. (b) Mitochondria and post-mitochondrial supernatants (cytosol) were isolated from strains expressing the mutant forms of Tob37 described in panel A. Samples of each were subjected to SDS-PAGE and transferred to nitrocellulose. The membrane was immunodecorated with the antibodies indicated on the left. The control was strain 76-26. Arginase represents a cytosolically localized control protein that is synthesized from two different start codons of the same locus [67] so that two bands of 41 kDa and 36 kDa are observed. (c) As in panel A except isolated mitochondria were treated for 30 min on ice with isolation buffer containing 0.5 M NaCl. Following the incubation period, mitochondria were pelleted. The supernatant was collected and desalted. The mitochondria were washed in isolation buffer and pelleted. Pelleted mitochondria and the desalted supernatant were subjected to SDS-PAGE. Proteins were transferred to nitrocellulose and the membrane was probed with the antibodies indicated on the left. (d) Mitochondria from the strains indicated above the panel were subjected to alkali treatment using 0.1 M sodium carbonate at pH 11.0, 11.5, and 12.5 (indicated below each panel) as described in the legend to Figure 4B. (e) Mitochondria were isolated from each of the Tob37 deletion protein strains and treated with proteinase K as described in the Materials and Methods and Figure 4A. (f) The large circle represents the cytosolic domain of Tob37, the filled box is TMD1, and the open box is the CHD. Two horizontal lines represent the mitochondrial outer membrane. The predicted arrangement of the domains for wild type and each of the TMD/CHD deletions is indicated. (g) Comparison of potential tail-anchoring sequences of N. crassa (Nc) Tob37 and H. sapiens (Hs) Mtx1. The potential TMD is indicated by the solid line. The position of the region within each protein is indicated by the numbers flanking each amino acid sequence. The overall length of the N. crassa protein is 442 residues. The H. sapiens protein is 304 residues. Positively charged residues in the immediate flanking regions are indicated by the plus sign.
Figure 6.
Characteristics of Tob37 C-terminal deletion strains.
(a) Conidia from the strains indicated on the left were plated as described in the legend to Figure 1B. (b) Mitochondria were isolated from the strains indicated at the top of the panel and analyzed as described in the legend to Figure 1C. The control was strain 76–26. (c) Mitochondria isolated from the strains indicated at the top of the panel were dissolved in 1% digitonin, subjected to BNGE, transferred to PVDF, and decorated with antibody to Tob55. The position of molecular weight markers is indicated on the left.
Figure 7.
Role of Tob37 predicted TMDs on the import of mitochondrial precursor proteins into mitochondria.
Import and assembly were analyzed as described in the legend to Figure 2 for the indicated precursors. (a) Import of F1β and AAC. (b) Assembly of Tom40. (c) Assembly of porin. (d) Assembly of Tom22.
Figure 8.
Hypothetical model for effects of Tob37 alterations on the TOB complex.
Tob55 is shown as a pore-containing light grey ring embedded in the membrane. Tob37, is represented as in Fig. 5F with TMD1 and CHD in the outer membrane and intermembrane space, respectively. Tob38 is shown in dark grey with a domain extending into the pore of Tob55. A. The normal TOB complex. B. CHD absent. Loss of CHD has no effect on porin assembly and mild effects on Tom40 assembly. All members of the complex are shown in their normal configuration C. TMD1 and CHD absent. This results in reduced accumulation of the porin precursor at the TOB complex. Effects on Tom40 are mild. A conformational change in Tob55 is shown as one possible effect caused by loss of TMD1 resulting in porin assembly defects. D. Only TMD1 absent. This results in reduced accumulation of both porin and Tom40 precursors at the TOB complex. The conformational change in Tob55 due to lack of TMD1 is shown as in C. However, an additional change due to the suggested mislocalization of the CHD, is represented as an effect on Tob38.