Fig 1.
Detection of protein N-myristoylation of human SAMM50 and TOMM40 by cell-free and cellular metabolic labeling.
A. Interspecies alignment of the N-terminal sequences of SAMM50 and TOMM40. N-myristoylation motifs are shown in grey in the N-terminal sequences. B. The gene products of cDNAs encoding SAMM50-FLAG, SAMM50-G2A-FLAG, TOMM40-FLAG, and TOMM40-G2A-FLAG were synthesized using an insect cell-free protein synthesis system in the presence of [3H]leucine or [3H]myristic acid. The labeled translation products were analyzed by SDS-PAGE and fluorography, as described in the Materials and methods. C. cDNAs encoding SAMM50-FLAG, SAMM50-G2A-FLAG, TOMM40-FLAG, and TOMM40-G2A-FLAG were transfected into COS-1 cells. The expression of proteins was evaluated by western blotting analysis using an anti-FLAG antibody. Protein N-myristoylation was evaluated by metabolic labeling with a myristic acid analog followed by click chemistry, as described in the Materials and methods.
Fig 2.
Protein N-myristoylation of SAMM50 but not of TOMM40 is required for proper targeting to mitochondria.
A. Intracellular localization of SAMM50 and SAMM50-G2A was determined by immunofluorescence staining of COS-1 cells transfected with cDNAs encoding these two proteins using an anti-SAMM50 antibody. Mitochondria were detected using MitoTracker Red. The same experiment was performed using non-transfected COS-1 cells as a control. B. Intracellular localization of TOMM40 and TOMM40-G2A was determined by immunofluorescence staining of COS-1 cells transfected with cDNAs encoding these two proteins using an anti-TOMM40 antibody. Mitochondria were detected using MitoTracker Red. The same experiment was performed using non-transfected COS-1 cells as a control.
Fig 3.
Protein N-myristoylation of SAMM50 but not of TOMM40 is required for binding of protein to the membrane.
Total cell extracts of non-transfected COS-1 cells [A] and COS-1 cells transfected with cDNA encoding tag-free SAMM50 [B] or TOMM40 [C] were separated into cytosolic and membrane fractions by using membrane protein extraction kit (Trident). Presence of SAMM50 and TOMM40 in each fraction was determined by western blotting analysis using anti-SAMM50 or anti-TOMM40 antibody, respectively. For this analysis, anti-GAPDH antibody was used to detect GAPDH, a cytoslic marker protein [A]. For the analysis of COS-1 cells transfected with cDNAs coding for SAMM50 [B] and TOMM40 [C], relative amount of protein (%) reside in the cytosolic and total membrane fraction was calculated from densitometric analysis as described in materials and methods. Data are expressed as mean ± SD for three independent experiments. *P < 0.005 vs. wild-type.
Fig 4.
Endogenous SAMM50 and TOMM40 expressed in mammalian cells are N-myristoylated.
A. Metabolic labeling of endogenous proteins expressed in COS-1 cells with [3H]myristic acid followed by immunoprecipitation with specific antibodies against SAMM50 and TOMM40 was performed. The labeled proteins were separated by SDS-PAGE and then detected by fluorography. B. Expression of endogenous SAMM50, TOMM40, and MIC19 in COS-1 cells was determined by western blotting analysis using the respective specific antibodies. For this analysis, tag-free-SAMM50, tag-free-TOMM40, and tag-free-MIC19 exogenously expressed in COS-1 cells were used as controls.
Fig 5.
Identification of MIC19 as a major N-myristoylated binding partner of SAMM50.
A. Metabolic labeling of N-myristoylated proteins expressed in COS-1 cells with [3H]myristic acid followed by immunoprecipitation with specific antibodies against SAMM50, TOMM40, and MIC19 was performed. The labeled proteins were separated by SDS-PAGE and then detected by fluorography (lanes 4–6). For this analysis, tag-free-SAMM50, -TOMM40, and -MIC19 exogenously expressed in COS-1 cells were used as controls (lanes 1–3). B. Western blotting analysis of immunoprecipitated samples used in A. Presence of endogenous SAMM50, TOMM40, and MIC19 in the immunoprecipitated samples used in A lanes 4–6 was determined by western blotting analysis. For this analysis, total cell lysates of COS-1 cells were used as control (lanes 1, 5, and 9). Arrowheads in B indicate the position of heavy- and light-chains of IgG used for immunoprecipitation.
Fig 6.
Detection of protein N-myristoylation occurring on human MIC19 and MIC25 by cellular metabolic labeling.
A. Interspecies alignment of the N-terminal sequences of MIC19 and MIC25. N-myristoylation motifs are shown in grey in the N-terminal sequences. B. cDNAs encoding MIC19-FLAG, MIC25-FLAG, and their G2A mutants were transfected into COS-1 cells. The expression of proteins was evaluated by western blotting analysis using anti-FLAG antibodies. Protein N-myristoylation was evaluated by metabolic labeling with a myristic acid analog followed by click chemistry, as described in the Materials and methods.
Fig 7.
Protein N-myristoylation of MIC19 but not of MIC25 is required for proper targeting to mitochondria.
A. Intracellular localization of MIC19 and MIC19-G2A was determined by immunofluorescence staining of COS-1 cells transfected with cDNAs encoding these two proteins using an anti-MIC19 antibody. Mitochondria were detected using MitoTracker Red. The same experiment was performed using non-transfected COS-1 cells as a control. B. Intracellular localization of MIC25 and MIC25-G2A was determined by immunofluorescence staining of COS-1 cells transfected with cDNAs encoding these two proteins using an anti-MIC25 antibody. Mitochondria were detected using MitoTracker Red. The same experiment was performed using non-transfected COS-1 cells as a control.
Fig 8.
Protein N-myristoylation of MIC19 but not of MIC25 is required for binding of protein to the membrane.
Total cell extracts of non-transfected COS-1 cells [A] and COS-1 cells transfected with cDNA encoding tag-free MIC19 [B] or MIC25 [C] were separated into cytosolic and membrane fractions by using membrane protein extraction kit (Trident). Presence of MIC19 and MIC25 in each fraction was determined by western blotting analysis using anti-MIC19 or anti-MIC25 antibody, respectively. For this analysis, anti-GAPDH antibody was used to detect GAPDH, a cytoslic marker protein [A]. For the analysis of COS-1 cells transfected with cDNAs coding for MIC19 [B] and MIC25 [C], relative amount of protein (%) reside in the cytosolic and total membrane fraction was calculated from densitometric analysis as described in materials and methods. Data are expressed as mean ± SD for three independent experiments. *P < 0.005 vs. wild-type.
Fig 9.
Expression and N-myristoylation of endogenous MIC19 and MIC25 in COS-1 cells.
A. Metabolic labeling of endogenous proteins expressed in COS-1 cells with [3H]myristic acid followed by immunoprecipitation with specific antibodies against MIC19 and MIC25 was performed. The labeled proteins were separated by SDS-PAGE and then detected by fluorography. B. Comparison of the molecular size of endogenous N-myristoylated MIC19 and MIC25 immunoprecipitated from COS-1 cells with that of N-myristoylated tag-free MIC19 and MIC25 exogenously expressed in COS-1 cells. [3H]myristic acid labeling of COS-1 cells transfected with cDNA coding for tag-free MIC19 or MIC25 followed by immunoprecipitation with specific antibodies against these two proteins was performed. The immunoprecipitated samples and the samples obtained in A were separated by SDS-PAGE and then detected by fluorography. C. Comparison of the relative amounts of MIC19 and MIC25 expressed in COS-1 cells. The expression level of endogenous MIC25 in COS-1 cells was compared with that of MIC19 by western blotting analysis using MIC19-FLAG and MIC25-FLAG as standard proteins. For this analysis, total cell lysates derived from non-transfected COS-1 cells or COS-1 cells transfected with cDNAs coding for MIC19-FLAG, tag-free MIC19, MIC25-FLAG, or tag-free MIC25 were subjected to western blotting analysis using anti-FLAG, anti-MIC19, or anti-MIC25 antibodies.
Fig 10.
Comparison of the relative amounts of MIC19 and MIC25 expressed in mammalian cells.
The expression levels of endogenous MIC25 in four mammalian cell lines (COS-1, HEK293T, HeLa, HepG2 cells) were compared with those of MIC19 by western blotting analysis using tag-free MIC19 and tag-free MIC25 expressed in COS-1 cells as standard proteins. For this analysis, total cell lysates derived from the four cell lines were subjected to western blotting analysis using anti-MIC19 or anti-MIC25 antibodies. A; Expression of MIC19, B; Expression of MIC25, Upper panels; Coomassie-brilliant blue (CBB) staining, Lower panels; Western blotting analysis.
Fig 11.
Protein N-myristoylation of MIC19 is required for the association of MIC19 with SAMM50.
Association of MIC19-FLAG stably expressed in COS-1 cells with endogenously expressed SAMM50 was studied by immunoprecipitation analysis. A. Expression of MIC19-FLAG and MIC19-G2A-FLAG in respective stable transformants. Upper panel; Western blotting analysis using an anti-FLAG antibody, Lower panel; Western blotting analysis using an anti-MIC19 antibody. B. Detection of protein N-myristoylation of MIC19-FLAG expressed in stable transformants of MIC19-FLAG. Upper panel; Western blotting analysis using an anti-FLAG antibody. Lower panel; Detection of protein N-myristoylation by metabolic labeling with myristic acid analog followed by detection with click chemistry. C. Western blotting analysis of MIC19-FLAG or MIC19-G2A-FLAG immunoprecipitated from stable transformants of MIC19-FLAG and MIC19-G2A-FLAG using an anti-MIC19 antibody. D. Analysis of the binding of MIC19-FLAG or MIC19-G2A-FLAG to endogenous SAMM50 by western blotting analysis of immunoprecipitated samples of stable transformants of MIC19-FLAG and MIC19-G2A-FLAG using an anti-SAMM50 antibody. The experiments were performed in triplicate. A typical pattern of western blotting is presented. E. Quantitative analysis of MIC19-FLAG or MIC19-G2A-FLAG bound to SAMM50 was performed using the results obtained in D. Data are expressed as mean ± SD for three independent experiments. *P < 0.01 vs. wild-type.
Fig 12.
Schematic representation of the intracellular localization and protein-protein interactions of SAMM50, TOMM40, and MIC19.
SAMM50, TOMM40, MIC19, and MIC25 were cotranslationally N-myristoylated in the cytoplasm. These proteins are transported into the intermembrane space of mitochondria through the TOM complex. For SAMM50 and MIC19, protein N-myristoylation is required for mitochondrial targeting. An oligomeric form of N-myristoylated MIC19 seems to associate with N-myristoylated SAMM50. Protein N-myristoylation of MIC19 is required for the binding of MIC19 to SAMM50.