Genetic and functional interactions between the mitochondrial outer membrane proteins Tom6 and Sam37

Abstract

The TOM complex is the general mitochondrial entry site for newly synthesized proteins. Precursors of beta-barrel proteins initially follow this common pathway and are then relayed to the SAM/TOB complex, which mediates their integration into the outer membrane. Three proteins, Sam50 (Tob55), Sam35 (Tob38/Tom38), and Sam37 (Mas37), have been identified as the core constituents of the latter complex. Sam37 is essential for growth at elevated temperatures, but the function of the protein is currently unresolved. To identify interacting partners of Sam37 and thus shed light on its function, we screened for multicopy suppressors of sam37Delta. We identified the small subunit of the TOM complex, Tom6, as such a suppressor and found a tight genetic interaction between the two proteins. Overexpression of SAM37 suppresses the growth phenotype of tom6Delta, and cells lacking both genes are not viable. The ability of large amounts of Tom6 to suppress the sam37Delta phenotype can be linked to the capacity of Tom6 to stabilize Tom40, an essential beta-barrel protein which is the central component of the TOM complex. Our results suggest that Sam37 is required for growth at higher temperatures, since it enhances the biogenesis of Tom40, and this requirement can be overruled by improved stability of newly synthesized Tom40 molecules.

FIG. 1.

TOM6 is a high-copy-number suppressor of sam37Δ. (A) Cells lacking Sam37 were transformed with a plasmid containing SAM37, plasmids isolated from suppressor colonies 3 and 4, or an empty plasmid (−). The cells were tested by drop dilution assay for their ability to grow at 37°C on the indicated medium. (B) Sam37Δ cells harboring plasmids expressing the indicated genes or an empty plasmid (−) were tested by drop dilution assay for their ability to grow at 37°C on the indicated medium. (C) Expression of TOM6-HA from a multicopy plasmid results in elevated amounts of the protein. Crude mitochondria were isolated from wild-type (WT) cells transformed with an empty plasmid, cells where the chromosomal copy of TOM6 was replaced by TOM6-HA, and from wild-type and sam37Δ cells transformed with a multicopy plasmid containing TOM6-HA. All cells were grown at 30°C. Equal amounts were analyzed by sodium dodecyl sulfate (SDS)-PAGE and immunodecoration with an antibody against either the HA tag or the outer membrane protein Tom70 as a control. (D) The multicopy SAM37 plasmid results in overproduction of Sam37. Sam37Δ cells were transformed with either an empty vector (pRS426) or a plasmid carrying TOM6 or SAM37. Mitochondria were isolated from wild-type cells and the transformed cells, and the indicated amounts of the organelles were analyzed by SDS-PAGE and immunodecoration with the indicated antibodies. Dld1 is anchored to the inner membrane and serves here as a control protein.

FIG. 2.

Receptor subunits of the TOM complex and the small Tim chaperones cannot rescue the sam37Δ growth phenotype. (A) sam37Δ cells transformed with either an empty pRS426 vector (−) or a plasmid encoding the indicated Tom subunit were used. Cells were grown at 30°C and ruptured by vortexing in the presence of glass beads. Crude mitochondria were obtained by differential centrifugation and analyzed by sodium dodecyl sulfate-PAGE. Proteins were blotted onto a membrane, which was then immunodecorated with antibodies (indicated by “α”) against the indicated Tom subunit or with antibodies against the matrix protein Hep1. (B) sam37Δ cells harboring plasmids expressing the indicated genes or an empty vector (−) were tested by drop dilution assay for their ability to grow at 37°C on the indicated medium.

FIG. 3.

Overexpression of SAM37 can rescue a tom6Δ growth defect. Cells devoid of TOM6 were transformed with a plasmid carrying the indicated gene or with an empty plasmid (−). The transformed cells were tested by drop dilution assay for their ability to grow at the indicated temperatures on glycerol-containing synthetic medium (SG-Ura).

FIG. 4.

Double deletion of SAM37 and TOM6 results in synthetic lethality. Wild-type (WT) and sam37Δ tom6Δ cells containing plasmid-borne SAM37 or TOM6 were plated at 30°C on glucose-containing rich medium (YPD) (A) or 5-FOA-containing medium (5-FOA medium) (B). Wild-type (WT) and sam37Δ tom7Δ cells were plated in a similar manner (C and D). The pRS426 vector used in this experiment contains a URA3 gene, which upon its expression converts the nontoxic 5-FOA compound to toxic 5-fluorouracil. Thus, a loss of this plasmid is favored under these conditions.

FIG. 5.

Expression levels of TOM6 do not affect the SAM complex. (A) Mitochondria were isolated from wild-type (WT) or tom6Δ cells transformed with an empty vector (pRS426) or from wild-type cells transformed with a plasmid carrying TOM6. The given amounts of mitochondria were analyzed by sodium dodecyl sulfate (SDS)-PAGE and immunodecoration with antibodies against the indicated mitochondrial outer membrane proteins. (B) The indicated amounts of mitochondria were analyzed by BN-PAGE and immunodecoration with antibodies against Sam50. (C) Mitochondria isolated as described above were incubated with radiolabeled precursor of porin at 25°C for various time periods. Samples were then treated with proteinase K, and mitochondria were reisolated. Imported porin was analyzed by SDS-PAGE and autoradiography.

FIG. 6.

Overexpression of TOM6 stabilizes Tom40. (A) Wild-type (WT) cells transformed with an empty vector (pRS426) and sam37Δ cells transformed with either an empty vector (pRS426) or a plasmid carrying TOM6 or TOM7 were used. Cells were grown at 30°C and ruptured by vortexing in the presence of glass beads. Crude mitochondria were obtained by differential centrifugation (M) and were analyzed together with the postmitochondrial supernatant fraction (P) by sodium dodecyl sulfate (SDS)-PAGE. Proteins were blotted onto a membrane, which was then immunodecorated with antibodies against the indicated mitochondrial outer membrane proteins or with antibodies against the cytosolic marker protein Bmh1. The results, for each protein, of at least three experiments were quantified, and the average values are presented. Error bars represent standard deviations. For each protein, the amount in wild-type mitochondria was set at 100%. (B) Mitochondria were isolated from cells grown at 32°C. The indicated amounts of mitochondria were analyzed by SDS-PAGE and immunodecoration with antibodies against the indicated mitochondrial outer membrane proteins. The intensities of the bands corresponding to the various proteins were quantified. For each amount of mitochondria, the intensity of the bands in the altered mitochondria was calculated as a percentage of the signal obtained from the wild-type organelle. For each protein, the average value of the two different amounts of organelles from at least three experiments is presented. Error bars represent standard deviations.

FIG. 7.

Overexpression of TOM6 stabilizes the TOM complex (A). Mitochondria were isolated from cells grown at 30°C and solubilized in 1% digitonin before their analysis by BN-PAGE and immunodecoration with antibodies (indicated by “α”) against either Tom40 or Tom70 (as a control). The migration behavior of molecular mass markers and bands corresponding to assembled TOM complex are indicated. Of note, employing two different amounts of mitochondria resulted in two different ratios of detergent/protein. This difference is reflected in a minor change in the migration behavior of the complexes. The bands corresponding to the assembled TOM complex in samples containing 80 μg mitochondria were quantified. The amount in wild-type (WT) organelle was set to 100%, and the average values of at least three experiments are presented. Error bars represent standard deviations. (B) Mitochondria were isolated from cells grown at 32°C and analyzed by BN-PAGE as in part A. The bands corresponding to the assembled TOM complex or dimeric Tom70 (as a control) in samples containing 80 μg mitochondria were quantified. The amount in wild-type organelle was set to 100%, and the average values for at least three experiments are presented. Error bars represent standard deviations. (C) Mitochondria at the indicated amounts were solubilized in 0.5% Triton X-100 and analyzed by BN-PAGE and immunodecoration with antibodies against Sam50. The migration behavior of molecular mass markers and bands corresponding to either SAM or Sam50-Sam35 core complexes are indicated.

FIG. 8.

An overexpression plasmid encoding Tom40 cannot suppress the growth phenotype of sam37Δ cells. (A) Crude mitochondria were isolated from both wild-type (WT) and sam37Δ cells transformed with either an empty vector (pRS426) or a plasmid carrying TOM40. Cells were grown at 30°C. Equal amounts of mitochondria were analyzed by sodium dodecyl sulfate-PAGE and immunodecoration with antibodies against either Tom40 or Tom70. (B) Wild-type and sam37Δ cells harboring an empty pRS426 and sam37Δ cells transformed with a plasmid carrying TOM40 were tested by drop dilution assay for their ability to grow at 37°C on synthetic medium lacking Ura.

FIG. 9.

Overexpression of TOM6 suppresses the Tom40 import phenotype of sam37Δ cells. (A) Mitochondria were isolated from wild-type (WT) and sam37Δ cells harboring an empty vector and from sam37Δ cells overexpressing TOM6. Mitochondria were incubated with radiolabeled precursor of Tom40 at 25°C for various time periods. Upper panel: samples were treated with proteinase K (PK), and mitochondria were reisolated. Imported Tom40 was analyzed by sodium dodecyl sulfate (SDS)-PAGE and autoradiography. Lower panel: mitochondria were solubilized with 1% digitonin, and samples were analyzed by BN-PAGE and autoradiography. Assembly intermediates I and II and the assembled TOM complex are indicated. An unspecific band is marked with an asterisk. The bands corresponding to PK-protected Tom40 (import) and assembled Tom40 (assembly) were quantified, and an experiment representative of three independent experiments is presented. The amount of protein imported into or assembled within wild-type mitochondria after the longest incubation period was set to 100%. (B) Mitochondria as for part A were incubated with radiolabeled precursor of porin at 25°C for various time periods. Further treatment and analysis were as described in the legend to part A. The bands corresponding to assembled porin are indicated. The bands corresponding to the PK-protected and the assembled porin were quantified. (C) Mitochondria as for part A were incubated with radiolabeled precursor of Sam50 at 25°C for various time periods. Samples were treated with PK, and mitochondria were reisolated. Imported Sam50 was analyzed by SDS-PAGE and autoradiography. Two characteristic proteolytic fragments with apparent molecular masses of 30 and 25 kDa are indicated with F′ and F″, respectively (see reference 15). The bands corresponding to F′ were quantified, and the intensity of this fragment formed upon import into mitochondria isolated from wild-type cells for the longest time period was set to 100%. An experiment representative of three independent experiments is presented.

FIG. 10.

Overproduction of Tom6 suppresses Tom22 assembly and improves import phenotypes of sam37Δ cells. (A) Radiolabeled precursor of Tom22 was incubated with various mitochondria as described in the legend to Fig. 9A. Either samples were extracted with carbonate and pellets were analyzed by sodium dodecyl sulfate (SDS)-PAGE and autoradiography, or they were solubilized with 1% digitonin and analyzed by BN-PAGE and autoradiography. Bands corresponding to assembled TOM complex are indicated. The bands corresponding to Tom22 in the carbonate pellet (import) and assembled Tom22 (assembly, assayed by BN-PAGE) were quantified. The amount of protein imported into or assembled within mitochondria isolated from wild-type (WT) cells after the longest incubation period was set to 100%. An average for three experiments is presented. (B) Radiolabeled precursor of pSu9-DHFR was incubated with various mitochondria as described in the legend to Fig. 9A. Samples were treated with proteinase K, mitochondria were reisolated, and imported proteins were analyzed by SDS-PAGE and autoradiography. The precursor and mature forms are indicated by p and m, respectively. The bands corresponding to the mature form were quantified, and the amount of protein imported into mitochondria isolated from wild-type cells for the longest time period was set to 100%.

FIG. 11.

Large amounts of Tom6 suppress the morphology phenotype of sam37Δ cells. (A) Wild-type cells (wt), sam37Δ cells, and sam37Δ cells overexpressing TOM6 were transformed with a vector expressing mitochondrially localized GFP. Cells were grown at 32°C on liquid glucose-containing medium and were observed by fluorescence microscopy. Mitochondrial morphology (mitoGFP) and bright-field images are presented (bar, 5 μm). (B) Quantification of the phenotypes of at least 100 cells from each strain. Cells were grown at 30°C and classified into those containing normal tubular (normal), condensed, or fragmented mitochondria. WT, wild type. (C) Cells grown at 25°C harbor reduced levels of Tom40 and porin. Crude mitochondria were isolated from both wild-type (WT) and sam37Δ cells transformed with an empty vector (pRS426) and from sam37Δ cells overexpressing TOM6. Equal amounts of mitochondria were analyzed by sodium dodecyl sulfate-PAGE and immunodecoration with antibodies against the indicated mitochondrial proteins. The bands corresponding to Tom40 and Tom70 from three experiments were quantified. The amount in wild-type mitochondria was set to 100%. Error bars represent standard deviations. (D) Cells as in panel A were grown at 25°C. For each strain, more than 100 cells were analyzed by fluorescence microscopy for their mitochondrial morphology. The percentage of cells containing normal tube-like mitochondria is presented. WT, wild type.

FIG. 12.

In vitro import of precursor proteins is not reduced in mitochondria isolated from fis1Δ cells. Mitochondria were isolated from wild-type (WT) and fis1Δ cells. Organelles were incubated with the radiolabeled precursors of Tom40, porin, or pSu9-DHFR at 25°C for various time periods. Samples were treated with proteinase K, and mitochondria were reisolated. Imported proteins were analyzed by sodium dodecyl sulfate-PAGE and autoradiography. The precursor and mature forms of pSu9-DHFR are indicated as p and m, respectively.

FIG. 13.

Overexpression of SAM37 in tom6Δ cells does not stabilize the endogenous TOM complex but improves the assembly of newly synthesized Tom40 precursor molecules. (A) Mitochondria were isolated from the indicated strains, solubilized in 1% digitonin, and analyzed by BN-PAGE and immunodecoration with antibodies against Tom40. The migration behavior of molecular mass markers and bands corresponding to assembled and dissociated (diss.) TOM complex are indicated. WT, wild type. (B) Mitochondria isolated as described above were incubated with radiolabeled precursor of Tom40 at 25°C for various time periods. Mitochondria were reisolated and solubilized with 1% digitonin, and samples were analyzed by BN-PAGE and autoradiography. Assembly intermediates I and II and the assembled TOM complex are indicated. An unspecific band is marked with an asterisk. The bands corresponding to assembled Tom40 for three independent experiments were quantified, and a representative experiment is presented. The amount of protein assembled within wild-type mitochondria after the longest incubation period was set to 100%.