Cytosolic signaling protein Ecsit also localizes to mitochondria where it interacts with chaperone NDUFAF1 and functions in complex I assembly

Abstract

Ecsit is a cytosolic adaptor protein essential for inflammatory response and embryonic development via the Toll-like and BMP (bone morphogenetic protein) signal transduction pathways, respectively. Here, we demonstrate a mitochondrial function for Ecsit (an evolutionary conserved signaling intermediate in Toll pathways) in the assembly of mitochondrial complex I (NADH:ubiquinone oxidoreductase). An N-terminal targeting signal directs Ecsit to mitochondria, where it interacts with assembly chaperone NDUFAF1 in 500- to 850-kDa complexes as demonstrated by affinity purification and vice versa RNA interference (RNAi) knockdowns. In addition, Ecsit knockdown results in severely impaired complex I assembly and disturbed mitochondrial function. These findings support a function for Ecsit in the assembly or stability of mitochondrial complex I, possibly linking assembly of oxidative phosphorylation complexes to inflammatory response and embryonic development.

Figure 1.

Ecsit isoform 1 copurifies with tandem affinity-purified NDUFAF1. (A) Ten Ecsit splice isoforms are predicted by the EBI alternative splicing database, of which two are identified in experimental studies (isoforms 1 and 2). Shown are the exons (vertical bars) and introns (horizontal lines) that comprise the complete transcript. The Swissprot annotation and predicted molecular mass are indicated when available. (B) FT-MS sequence coverage of Ecsit in eluates of tandem affinity-purified NDUFAF1 in HEK293 mitochondria demonstrates the presence of Ecsit isoform 1. Identified peptides are indicated in bold and in boxes, whereas exon 6, unique for Ecsit isoform 1, is indicated in gray.

Figure 2.

Ecsit localizes to mitochondria and interacts with NDUFAF1 in three high-molecular-weight complexes. (A) HEK293 cells were fractionated by pottering and lysates from total cell; mitochondria and cytoplasm were subsequently analyzed by SDS-PAGE and Western blotting. (T) Total cell; (C) cytoplasm; (M) mitochondria. All lanes were immunodecorated with antibodies targeted to Ecsit, NDUFAF1, TRAF6, cytoplasmic control GAPDH, and mitochondrial control COXII. Ecsit is predominantly present in the cytoplasm; however, a smaller band of ∼45 kDa is visible specifically in mitochondria. In contrast, the cytoplasmic Ecsit-binding partner TRAF6 is not detected in mitochondria. (B) Trypsin import assay. (T) Total cell; (C) cytoplasm; (M) mitochondria; (M +T) mitochondria + 30 U trypsin 15 min at 37°C; (M ++T) same as M +T but with 100 U trypsin; (M +T lysis) same as M +T but with 1% Triton to lyse the mitochondria. Increasingly stringent trypsin digestion of mitochondrial outer membrane proteins results in the disappearance of the 50-kDa Ecsit, whereas the 45-kDa Ecsit remains intact. NDUFS3 is used as a mitochondrial matrix control, Tom20 is used as an outer membrane control. (C) Immunoprecipitation using an anti-myc antibody in mitochondria purified from an NDUFAF1-myc-HIS-inducible HEK293 cell line. Shown are mitochondria (M), nonbound (NB), and eluate fractions (E). Myc-immunoprecipitation coelutes the 45-kDa mitochondrial Ecsit together with NDUFAF1, as opposed to mitochondrial controls ND1, NDUFA1, and COXII, and cytoplasmic control TRAF6. (D) Two-dimensional blue-native SDS-PAGE analysis of HeLa and HEK293 mitochondrial lysates demonstrates the colocalization of 45-kDa Ecsit and NDUFAF1 in three complexes of ∼500, 600, and 850 kDa. Complex I subunit NDUFS3 is shown to demonstrate the position of complex I (“A,” 1 MDa). Subcomplexes observed in our previous complex I assembly study (Ugalde et al. 2004) are indicated with A–H when visible. (E) HEK293 mitochondrial Ecsit signal was compared with a total cell preparation. (50) The 50-kDa cytoplasmic Ecsit in the HEK293 total cell lysate, which is absent in HEK293-purified mitochondria. This demonstrates that only the 45-kDa, mitochondrial, Ecsit interacts with NDUFAF1 in complexes of 500–850 kDa.

Figure 3.

Ecsit requires its N-terminal targeting sequence for mitochondrial localization. (A–H) Confocal microscopy of HEK293 cells transiently transfected with an inducible Ecsit-GFP construct (A–D) and with an inducible Ecsit-GFP construct lacking the 48-amino-acid Ecsit N terminus (E–H). A and E show GFP signal. B and F show the mitochondrial network using Mitotracker Red. C and G show nuclear staining. D and H show the overlay between the three signals. Bars, 10 μm. Without the N terminus, Ecsit-GFP is no longer targeted to mitochondria. Thus, Ecsit-GFP requires its N-terminal targeting sequence for mitochondrial localization. (I) Cell fractionation of Ecsit-GFP-inducible HEK293 cells. (+) Induction of expression; (−) no induction of expression; (T) total cell; (C) cytoplasm; (M) mitochondria. NDUFS3 is used as mitochondrial control. Other antibodies used are anti-Ecsit (“Ecsit” panel) and anti-GFP (“GFP” panel). The endogenous Ecsit signals are visible at 50 kDa (total cell and cytoplasm) and 45 kDa (mitochondria). The induced Ecsit-GFP targets predominantly to mitochondria and is detected at ∼70 kDa (45 kDa + 24 kDa) in two bands using the anti-Ecsit antibody. Only one of these anti-Ecsit-stained bands can be made visible using the anti-GFP antibody (“GFP” panel). The sensitive anti-GFP antibody also shows minor Ecsit-GFP leakage expression (in the uninduced situation). (J) Cell fractionation of inducible HEK293 cells expressing Ecsit-GFP without N terminus, as performed in I. In the induced situation, Ecsit appears predominantly in total cell and cytoplasm as several bands migrating at ∼70–75 kDa (“Ecsit” panel), of which only one is detected using the anti-GFP antibody (“GFP” panel). Again, minor leakage expression is observed in the uninduced situation. (K) Two-dimensional analysis of Ecsit-GFP incorporation into high-molecular-weight Ecsit/NDUFAF1 protein complexes of 500–850 kDa. The NDUFS3 signal is used as a marker for previously observed complex I subcomplexes A–H when visible (Ugalde et al. 2004). In accordance with A, two types of Ecsit-GFP are visible on the anti-Ecsit incubated blot (“Ecsit” panel), of which only one is detectable using anti-GFP antibody (“GFP” panel). The Ecsit-GFP complexes comigrate with the NDUFAF1 complexes (500–850 kDa). In addition, a larger complex matching the size of complex I (1 MDa) is indicated with an asterisk.

Figure 4.

Ecsit knockdown using RNAi results in disturbed complex I assembly. (A) RNAi was performed using two siRNAs (#1 and #2) against Ecsit mRNA. Following SDS-PAGE and Western blotting, immunodetection was performed for Ecsit, NDUFAF1, COXII, and NDUFS3 in untreated (U), mock-transfected (M), and siRNA-transfected (#1 and #2) HeLa cells. The Ecsit signal is knocked down, which correlates with a severe depression in NDUFAF1 protein. Monomeric COXII and NDUFS3 levels remain unchanged. (B) The effect of Ecsit knockdown on OXPHOS complex assembly was investigated by blue-native PAGE followed by Western blotting and immunodetection of complex I subunit NDUFS3 (CI), complex II subunit SDHA (CII), complex III subunit core2 (CIII), complex IV subunit COXII (CIV), and complex V subunit ATPase α (CV). Arrows indicate accumulated subcomplexes detected with the anti-NDUFS3 antibody. (C) Two-dimensional blue-native SDS-PAGE analysis of samples analyzed in A and B. Shown are immunodetections of Ecsit, NDUFAF1, and complex I (NDUFS3 and ND1 signals), in untreated, mock-transfected, and Ecsit siRNA-transfected (#1 and #2) HeLa cells. Subcomplexes that correspond to previously described complex I subcomplexes (Ugalde et al. 2004) are indicated with A–H. (Sub) Accumulated subcomplexes after both Ecsit siRNA transfections. The Ecsit/NDUFAF1 complexes are indicated with 500–850 kDa. An asterisk indicates signal from a previous NDUFS3 detection.

Figure 5.

Ecsit knockdown using RNAi affects mitochondrial and cellular physiology. (A) Complex I in-gel activity (CI-IGA) and complex II (CII) expression following native electrophoresis in untreated (U), mock-treated (M), and two Ecsit RNAi knockdowns (E#1 and E#2). The bottom panel depicts the complex I in-gel activity (CI-IGA) signals corrected for complex II (CII) expression (expressed as percentage of the value in untreated cells) determined by integrated optical density analysis. (B) NAD(P)H levels in untreated (U), mock-treated (M), and siRNA-treated (E#1 and E#2) cells. Bars represent the average of 262 (U), 293 (M), 234 (E#1), and 180 (E#2) cells. (C) Cellular superoxide (filled bars) and oxidant levels (open bars) in untreated (U), mock-treated (M), and siRNA-treated (E#1 and E#2) cells. Bars represent the average of 294 (U), 284 (M), 335 (E#1), and 93 (E#2) cells for superoxide levels, and 47 (U), 33 (M), 41 (E#1), and 25 (E#2) cells for oxidant levels. (D) Degree of mitochondrial branching (F, black bars) and number of mitochondria per cell (Nc, open bars) in untreated (U), mock-treated (M), and siRNA-treated (E#1 and E#2) cells. Bars represent the average of 50 (U), 56 (M), 70 (E#1), and 50 (E#2) cells. In B–D, letters (a, b, c, and d) represent statistically significant differences with the indicated columns. Data was obtained during two independent experiments from multiple cells (N).