The peripheral membrane subunits of the SAM complex function codependently in mitochondrial outer membrane biogenesis

Abstract

The sorting and assembly machinery (SAM) complex functions in the assembly of beta-barrel proteins into the mitochondrial outer membrane. It is related to the Omp85/YaeT machinery in bacterial outer membranes, but the eukaryotic SAM complex is distinguished by two peripheral subunits, Sam37 and Sam35, that sit on the cytosolic face of the complex. The function of these subunits in beta-barrel protein assembly is currently unclear. By screening a library of sam35 mutants, we show that 13 distinct alleles were each specifically suppressed by overexpression of SAM37. Two of these mutants, sam35-409 and sam35-424, show distinct phenotypes that enable us to distinguish the function of Sam35 from that of Sam37. Sam35 is required for the SAM complex to bind outer membrane substrate proteins: destabilization of Sam35 inhibits substrate binding by Sam50. Sam37 acts later than Sam35, apparently to assist release of substrates from the SAM complex. Very different environments surround bacteria and mitochondria, and we discuss the role of Sam35 and Sam37 in terms of the problems peculiar to mitochondrial protein substrates.

Figure 1.

Sam37 is a multicopy suppressor for sam35 alleles. (A) Δsam35 cells expressing wild-type SAM35 or sam35-424 from a plasmid and transformed with the indicated multicopy plasmids, were grown to mid-logarithmic phase. Five-fold serial dilutions were spotted onto synthetic complete medium (with glucose as a carbon source) lacking tryptophan and leucine, and plates were incubated at the indicated temperatures for 3 d. (B) Overexpression of Sam37 suppresses sam35-409 phenotype at restrictive temperature. Same as in A, except Δsam35 cells were transformed with plasmids encoding sam35-409 instead. (C) Sam37 and Sam35 are not functionally redundant. NCY0601 (heterozygous diploid SAM35/Δsam35) cells were transformed with a multicopy vector alone (YEplac195) or with the same vector containing SAM35 (YEpSam35) or SAM37 (YEpSam37). Transformed cells were sporulated and tetrads dissected on rich medium containing glucose. Results for four dissected asci are shown.

Figure 2.

Characterization of sam35-424. (A) Cells expressing wild-type SAM35 or sam35-424 were grown in lactate medium at 25°C, and mitochondria were isolated. Mitochondrial proteins were separated by SDS-PAGE, transferred onto nitrocellulose membrane, and immunodecorated with antisera against the indicated proteins. (B) Mitochondria (100 μg) from the indicated strains were solubilized in 1% digitonin, and protein complexes were separated by BN-PAGE and blotted onto PVDF membranes. Anti-Tom40 antisera were used to immunoblot for the TOM complex. (C) Mitochondria (100 μg) from the indicated strains were solubilized in 1% digitonin, and protein complexes were separated by BN-PAGE and blotted onto PVDF membranes. Antisera against Sam50 or Sam35 were used to immunoblot for the SAM complex. Asterisks, unidentified subcomplexes of the SAM complex obtained during solubilization; arrow, Sam50-containing complex specifically enriched in sam35-424. (D) Mitochondria from cells expressing wild-type SAM35 or sam35-424 were grown in lactate medium at 25°C, mitochondria were isolated and incubated at 25 or 37°C for 15 min in import buffer, followed by an equilibration at 25°C for 5 min. ³⁵S-labeled porin was added to the mitochondria and incubated at 25°C for the indicated time, treated with 50 μg/ml proteinase K to remove unimported precursors, and analyzed by SDS-PAGE and digital autoradiography. Arrow, altered proteinase K cleavage profile of porin upon import into sam35-424 mitochondria preincubated at 37°C for 15 min. Su9-DHFR (matrix), Aac1 (inner membrane) and Tim9 (intermembrane space) precursors were imported into mitochondria from wild-type or sam35-424 cells, with 15 min preincubation at 37°C. Quantification of the results is shown below. Filled boxes, SAM35; open boxes, sam35-424. (E) ³⁵S-labeled Tom40 was incubated with mitochondria for the indicated time. Mitochondria were reisolated and solubilized in 1% digitonin, and protein complexes were analyzed by BN-PAGE and digital autoradiography.

Figure 3.

Overexpression of Sam37 suppresses the phenotypes of sam35-424 by functioning as an assembly factor downstream of Sam35. (A) Mitochondria from Δsam35 cells transformed with plasmid containing wild-type SAM35 and vector YEplac181, sam35-424 transformed with the vector YEplac181, and sam35-424 expressing multiple copies of SAM37 from YEplac181 were analyzed by SDS-PAGE, and immunoblot analysis was completed using antisera against the indicated proteins. (B) Mitochondria (100 μg) from wild-type SAM35 or sam35-424 cells, transformed with the indicated plasmids, were solubilized in 1% digitonin buffer. Protein complexes analyzed by BN-PAGE, followed by blotting onto PVDF membrane and immunodecoration by using anti-Sam50 antisera. Asterisks, unidentified subcomplexes of the SAM complex obtained during solubilization; arrow, Sam50-containing complex specifically enriched in sam35-424. (C) Mitochondria from the indicated strains were preincubated at 37°C for 15 min, followed by equilibration at 25°C for 5 min before incubation with ³⁵S-labeled porin or Tom40 at 25°C for the indicated time. Unimported precursors were removed by treatment with 50 μg/ml proteinase K, and imported proteins were analyzed by SDS-PAGE and digital autoradiography. Quantification of the results shown on the right. Filled boxes, SAM35; open boxes, sam35-424; open triangles, sam35-424 overexpressing SAM37. (D) ³⁵S-labeled Tom40 was imported. into mitochondria from the indicated strains. Shown on the right is a longer exposure of a portion from the same gel.

Figure 4.

Overexpression of Sam37 suppresses the phenotype of sam35-409 by maintaining the level of sam35 in the SAM complex. (A) Mitochondria from Δsam35 cells transformed with plasmid encoding wild-type SAM35 and vector YEplac181, sam35-409 transformed with the vector YEplac181, and sam35-409 expressing multiple copies of SAM37 from YEplac181 were isolated from lactate medium at 25°C. The indicated amount of total protein (micrograms) was analyzed by SDS-PAGE, blotted onto nitrocellulose membrane, and immunodecorated using antisera against the indicated proteins. (B) Mitochondria (100 μg) were solubilized in 1% digitonin buffer, and protein complexes were analyzed by BN-PAGE, followed by blotting onto PVDF membrane and immunodecoration using anti-Sam50 antisera. Asterisks, unidentified subcomplexes of the SAM complex obtained during solubilization. (C) Mitochondria (100 μg) were subject to BN-PAGE and immunoblot analysis using anti-Tom40 antisera.

Figure 5.

Restoration of the TOM and SAM complexes in sam35-409 by overexpression of Sam37 cure import and assembly defects into various mitochondrial subcompartments. (A) ³⁵S-labeled porin, Su9-DHFR, and Tom40 were incubated with mitochondria isolated from Δsam35 cells transformed with plasmid encoding wild-type SAM35 and vector YEplac181, sam35-409 transformed with the vector YEplac181, and sam35-409 expressing multiple copies of SAM37 from YEplac181 for the indicated time at 25°C. Precursors not imported were removed by treatment with 50 μg/ml proteinase K and analyzed by SDS-PAGE, followed by digital autoradiography. Quantification of the results is shown below. Filled boxes, SAM35; open boxes, sam35-409; open triangles, sam35-409 overexpressing Sam37. (B) ³⁵S-labeled Tom40 precursor was incubated with the mitochondria from the indicated strains for the indicated time at 25°C. Mitochondria were reisolated and solubilized in 0.8% digitonin buffer. Insoluble materials were pelleted, and protein complexes were analyzed by BN-PAGE and digital autoradiography. Quantification of the signals from each assembly intermediate as a percentage of the total signal from 250-kDa, 100-kDa intermediates, and the TOM complex is shown below. Black bars, SAM35; white bars, sam35-409; gray bars, sam35-409–overexpressing Sam37.

Figure 6.

Highly reduced levels of Sam35 contribute to phenotypes of Δsam37 cells. (A) Serial fivefold dilutions of wild-type (W303) cells transformed with YEplac195, Δsam37 cells transformed with YEplac195, and Δsam37 cells transformed with YEpSam35 were spotted onto synthetic complete medium plates lacking uracil and incubated at the indicated temperatures for 2–4 d. (B) Cells from the three strains described in A were grown to mid-logarithmic phase in synthetic complete medium with lactate at 30°C and stained with Mitotracker Red; cells were analyzed by confocal microscopy. One hundred cells from each strain were randomly selected, and their mitochondrial morphology was recorded. “Normal” (filled) represents mitochondria that exist as reticulated network of tubules, “Abberant” (open) represents mitochondria that exist as condensed organelles, aggregated organelles, or both. (C) Mitochondria were isolated from the three strains described in A in lactate medium at 25°C and the indicated amount (micrograms of total protein) analyzed by SDS-PAGE, blotted on to nitrocellulose membranes, and immunodecorated using antisera against the indicated proteins. (D) Mitochondria (100 μg of protein) from the indicated strains were solubilized in 1% digitonin buffer. Protein complexes were analyzed by BN-PAGE and blotted onto PVDF membranes, followed by immunodecoration by using anti-Tom40 antisera. (E) ³⁵S-labeled Su9-DHFR and porin precursors were incubated with the indicated strains (described in A) for the indicated time at 25°C. Precursors not imported were removed by treatment with 50 μg/ml proteinase K followed by analysis by SDS-PAGE and digital autoradiography. Quantification of the results is shown on the right. Filled boxes, W303; open boxes, Δsam37; and open triangles, Δsam37-overexpressing Sam35.

Figure 7.

Sam35–Sam50 complex is sufficient for binding and assembly of β-barrel substrates. (A) Mitochondria (100 μg of total protein) isolated from wild-type (W303) cells transformed with YEplac195, Δsam37 cells transformed with YEplac195, and Δsam37 cells transformed with YEpSAM35 were solubilized in 1% digitonin buffer, and protein complexes were analyzed by BN-PAGE, blotted onto PVDF membranes, and immunodecorated with antisera against Sam50 and Sam35. (B) ³⁵S-labeled Tom40 precursor was incubated with mitochondria from the indicated strains for the indicated time at 25°C, and import was stopped by incubation on ice. Mitochondria were reisolated and solubilized with 0.8% digitonin buffer. Insoluble materials were pelleted, and protein complexes analyzed by BN-PAGE and digital autoradiography.

Figure 8.

Schematic representation of β-barrel protein assembly in bacteria, humans, and yeast. In bacteria, Omp85, together with factors facing the periplasm, is necessary and sufficient for assisting correct assembly of β-barrel into the bacterial outer membrane (Voulhoux et al., 2003; Ruiz et al., 2005; Wu et al., 2005). In humans, Metaxin 1 and Metaxin 2 are associated with the mitochondrial outer membrane as part of an uncharacterized ∼600-kDa complex (Kozjak-Pavlovic et al., 2007) and assist Sam50 in assembling β-barrel proteins into the outer membrane. In yeast, Sam35 and Sam37 associate directly with Sam50 to from the SAM_core complex. Sam35 assists Sam50 to bind β-barrel precursors, and Sam37 is important for clearance of β-barrel precursors from the SAM complex, thereby assisting assembly. Sam35 and Sam37 are important for stabilizing each other at the SAM complex, most likely via direct interactions.