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An Essential Role of Sam50 in the Protein Sorting and Assembly Machinery of the Mitochondrial Outer Membrane

Kozjak, Vera, Wiedemann, Nils, Milenkovic, Dusanka, Lohaus, Christiane, Meyer, Helmut E., Guiard, Bernard, Meisinger, Chris, Pfanner, Nikolaus·2003·Journal of Biological Chemistry
DOI 10.1074/jbc.c300442200
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FIG. 1 Identification of the mitochondrial outer membrane protein Sam50. A , samples 1 and 2 , mitochondria from wild-type ( WT ) and yeast cells expressing protein A-tagged Mas37 were lysed in digitonin-buffer. After IgG-chromatography, the SAM-complex was eluted by 0.5 M sodium acetate, pH 3.4, separated by SDS-PAGE and stained with Coomassie Brilliant Blue R-250. The indicated bands were identified by mass spectrometry. Samples 3 and 4 , mitochondria from cells expressing Sam50 with HA-tag were separated by BN-PAGE and the SAM complex was detected by Western blotting with antibodies directed against HA or Mas37. B , deduced primary structure of Sam50. Blue, predicted 𝛂 -helical segment; red, predicted 𝛃 -strands (also in eukaryotic Sam50 homologues) according to BetaTPred2 (34). C , Sam50 is an integral mitochondrial membrane protein. Mitochondria isolated from yeast expressing Sam50 with HA-tag were sonicated in the presence of 0.5 M NaCl or subjected to treatment at pH 11.5. Samples were left untreated or subjected to 100,000 × g centrifugation; total (T), supernatant (S), pellet (P). D , Sam50 is an outer membrane protein. Mitochondrial outer and inner membrane vesicles were separated on a sucrose gradient and fractions were analyzed by Western blotting.
FIG. 1 Identification of the mitochondrial outer membrane protein Sam50. A , samples 1 and 2 , mitochondria from wild-type ( WT ) and yeast cells expressing protein A-tagged Mas37 were lysed in digitonin-buffer. After IgG-chromatography, the SAM-complex was eluted by 0.5 M sodium acetate, pH 3.4, separated by SDS-PAGE and stained with Coomassie Brilliant Blue R-250. The indicated bands were identified by mass spectrometry. Samples 3 and 4 , mitochondria from cells expressing Sam50 with HA-tag were separated by BN-PAGE and the SAM complex was detected by Western blotting with antibodies directed against HA or Mas37. B , deduced primary structure of Sam50. Blue, predicted 𝛂 -helical segment; red, predicted 𝛃 -strands (also in eukaryotic Sam50 homologues) according to BetaTPred2 (34). C , Sam50 is an integral mitochondrial membrane protein. Mitochondria isolated from yeast expressing Sam50 with HA-tag were sonicated in the presence of 0.5 M NaCl or subjected to treatment at pH 11.5. Samples were left untreated or subjected to 100,000 × g centrifugation; total (T), supernatant (S), pellet (P). D , Sam50 is an outer membrane protein. Mitochondrial outer and inner membrane vesicles were separated on a sucrose gradient and fractions were analyzed by Western blotting.CC-BY · From the PDF
FIG. 2 sam50 yeast mutants are defective in assembly of Tom40. A , growth of wild-type ( WT ) yeast and sam50 mutants on YPD or YPG. B , protein levels of sam50-1 and sam50-3 mitochondria. Mitochondria ( 𝛍 g of protein) isolated from yeast cells grown at 23 ° C were separated by SDS-PAGE and analyzed by Western blotting. C , TOM complex and SAM complex analyzed by BNPAGE and Western blotting with antiTom40 or anti-Mas37, respectively. Mitochondria were preincubated for 15 min at 37 ° C. D , sam50-1 and sam50-3 mutant mitochondria are impaired in the assembly pathway of Tom40 precursor. Mitochondria were preincubated for 15 min at 37 ° C. Import of 35 S-labeled Tom40 was performed at 25 ° C for the indicated times. The reisolated mitochondria were analyzed by BN-PAGE and digital autoradiography.
FIG. 2 sam50 yeast mutants are defective in assembly of Tom40. A , growth of wild-type ( WT ) yeast and sam50 mutants on YPD or YPG. B , protein levels of sam50-1 and sam50-3 mitochondria. Mitochondria ( 𝛍 g of protein) isolated from yeast cells grown at 23 ° C were separated by SDS-PAGE and analyzed by Western blotting. C , TOM complex and SAM complex analyzed by BNPAGE and Western blotting with antiTom40 or anti-Mas37, respectively. Mitochondria were preincubated for 15 min at 37 ° C. D , sam50-1 and sam50-3 mutant mitochondria are impaired in the assembly pathway of Tom40 precursor. Mitochondria were preincubated for 15 min at 37 ° C. Import of 35 S-labeled Tom40 was performed at 25 ° C for the indicated times. The reisolated mitochondria were analyzed by BN-PAGE and digital autoradiography.CC-BY · From the PDF
FIG. 3 Functional Sam50 is required for the biogenesis of porin but not of matrix proteins. A , wild-type ( WT ) and sam50 mutant mitochondria were preincubated for 15 min at 37 ° C, incubated with 35 S-labeled porin at 25 ° C for the indicated times, and analyzed by BN-PAGE and digital autoradiography. B and C , preincubation and import were performed as described in the legend to A , using the precursor of the 𝛂 -subunit of mitochondrial processing peptidase and Su9-DHFR. After import, the mitochondria were treated with proteinase K and analyzed by SDS-PAGE. Where indicated, 𝚫 𝛙 was dissipated. p , precursor; i , intermediate; m , mature.
FIG. 3 Functional Sam50 is required for the biogenesis of porin but not of matrix proteins. A , wild-type ( WT ) and sam50 mutant mitochondria were preincubated for 15 min at 37 ° C, incubated with 35 S-labeled porin at 25 ° C for the indicated times, and analyzed by BN-PAGE and digital autoradiography. B and C , preincubation and import were performed as described in the legend to A , using the precursor of the 𝛂 -subunit of mitochondrial processing peptidase and Su9-DHFR. After import, the mitochondria were treated with proteinase K and analyzed by SDS-PAGE. Where indicated, 𝚫 𝛙 was dissipated. p , precursor; i , intermediate; m , mature.CC-BY · From the PDF

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Metadata

Authors
Kozjak, V., Wiedemann, N., Milenkovic, D., Lohaus, C., Meyer, H. E., Guiard, B., Meisinger, C., & Pfanner, N.
Venue
Journal of Biological Chemistry
Publisher
Elsevier BV
Published
2003-12
Volume
278
Issue
49
Pages
48520-48523
ISSN
0021-9258
DOI
10.1074/jbc.c300442200
Cited by
311
References
0
Language
en
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Kozjak, V., Wiedemann, N., Milenkovic, D., Lohaus, C., Meyer, H. E., Guiard, B., Meisinger, C., & Pfanner, N. (2003). An Essential Role of Sam50 in the Protein Sorting and Assembly Machinery of the Mitochondrial Outer Membrane. Journal of Biological Chemistry. https://doi.org/10.1074/jbc.c300442200