The Mitochondrial Acyl-carrier Protein Interaction Network Highlights Important Roles for LYRM Family Members in Complex I and Mitoribosome Assembly

Abstract

NDUFAB1 is the mitochondrial acyl carrier protein (ACP) essential for cell viability. Through its pantetheine-4'-phosphate post-translational modification, NDUFAB1 interacts with members of the leucine-tyrosine-arginine motif (LYRM) protein family. Although several LYRM proteins have been described to participate in a variety of defined processes, the functions of others remain either partially or entirely unknown. We profiled the interaction network of NDUFAB1 to reveal associations with 9 known LYRM proteins as well as more than 20 other proteins involved in mitochondrial respiratory chain complex and mitochondrial ribosome assembly. Subsequent knockout and interaction network studies in human cells revealed the LYRM member AltMiD51 to be important for optimal assembly of the large mitoribosome subunit, consistent with recent structural studies. In addition, we used proteomics coupled with topographical heat-mapping to reveal that knockout of LYRM2 impairs assembly of the NADH-dehydrogenase module of complex I, leading to defects in cellular respiration. Together, this work adds to the catalogue of functions executed by LYRM family of proteins in building mitochondrial complexes and emphasizes the common and essential role of NDUFAB1 as a protagonist in mitochondrial metabolism.

Keywords: Mitochondria function or biology; acyl-carrier protein; affinity proteomics; blue native polyacrylamide gel electrophoresis; complex I; protein complex analysis; protein structure; protein-protein interactions.

Graphical abstract

Fig. 1.

Determination of NDUFAB1 and yACP1 interactomes. Affinity-enrichment mass spectrometry (AE-MS) were performed on (A) NDUFAB1^Flag cells and (C) yACP1^Flag cells. Following imputation of missing values for the control experiments, log₂ transformed LFQ Intensities were submitted to a modified two-sided two-sample t test with significance determined by permutation-based FDR statistics (28). The negative logarithmic p values are plotted against the differences between the means for the two groups. A threshold of significance of < 1% FDR @ s0 = 5 for panel A and 2 for panel C was determined as described in the methods. Schematic representation of LYRM-family proteins interacting with (B) NDUFAB1 and (D) yACP1. Enriched proteins known to interact with LYRM proteins and their associated pathways are shown in the outer ring.

Fig. 2.

Proteomic and BN-PAGE analysis of select LYRM^KO lines. Volcano plots demonstrating relative levels of proteins in (A) LYRM2^KO, (B) AltMiD51^KO, (C) LYRM1^KO and (D) LYRM9^KO cells. The mean of log₂ transformed SILAC ratios for mitochondrial proteins detected for each KO against the HEK293T Control was analyzed by a single sample two-sided Student's t test and plotted against the negative log₁₀ p value. n = 3 biological replicates. Dot colors as indicated. E–I, Mitochondria were isolated from control and KO cell lines, solubilized in 1% digitonin, subjected to BN-PAGE and immunoblottng with antibodies for (E) NDUFA9 (complex I), (F) Core 1 (complex III), (G) COX4 (complex IV), (H) ATP5A (complex V) and (I) MRPL45 (mitoribosome large subunit, LSu). Blots were re-probed with complex II subunit SDHA as loading control. SC, supercomplex; SC^†, supercomplex assembly defect; CIII₂, complex III dimer, CIV, complex IV; mtRibo, mitoribosome; F₁, F₁-ATPase assemblies; asterisk, nonspecific complex; #, non-assembled COX4. (J) Mitochondrial complexes were quantified and shown as a percentage of control levels. SC(CI), SC(CIII₂), free CIII₂, CV and mitoribosomal complex (mitoRibo) represent signals from digitonin-solubilized samples whereas total CI, total CIII and total CIV represent signals from Triton X-100 solubilized samples. Data shown as mean ± S.E., n = 3.

Fig. 3.

Analysis of mitoribosome and translation defects in AltMiD51^KO cells. Topographical heat maps were fitted to the (A) mature mitoribosome (PDB: 3J9M (71)) and (B) stable assembly intermediate (PDB: 500L (19)). Blue is relatively less abundant, red is relatively more abundant, white is unchanged, dark gray is not detected and yellow is AltMiD51. rRNA is depicted in gold. C, Topographical heat-maps of the mitochondrial respiratory chain enzymes (complex I PDB: 5XTH (48), complex II PDB: 1ZOY (49), complex III dimer PDB: 1BGY (50), complex IV PDB: 5Z62 (72) and complex V PDB: 5ARE (52)). D, SDS-PAGE and phosphorimage analysis of ³⁵S-Met pulse labeled mtDNA subunits from control, AltMiD51^KO and AltMiD51^KO cells expressing AltMiD51^Flag. Immunoblotting using antibodies directed to the Flag epitope and SDHA were used as controls. E, Mitochondria were isolated from control, knockout and stably complemented cell lines, separated by BN-PAGE and assayed by Western blotting with antibodies against mitochondrial ribosome (MRPL45). CII serves as a loading control. F, As for E except probing for complex I (NDUFV1). LSu, large subunit; SSu, small subunit.

Fig. 4.

Defects in assembly of the complex I N-module in LYRM2^KO cells. A, Topographical heatmap of proteomic analysis of LYRM2^KO fitted to the complex I structure. B, Heatmap showing relative decrease in abundance of each complex I subunit. “X” denotes undetected protein. C–D, Mitochondria from control and LYRM2^KO cells were solubilized in Triton X-100, subjected to BN-PAGE and Western blot analysis using antibodies against NDUFA9 and NDUFAF2 respectively. E, Mitochondria were isolated from cells grown in the presence of chloramphenicol (CAP) for the time indicated, solubilized in digitonin and subjected to BN-PAGE and Western blotting using antibodies directed to NDUFA9 or NDUFAF2. TOM40 (detecting the TOM complex) was used as a loading control. F, Data in (E) was quantified and shown relative to “0 h” for each sample. Data shown as mean ± S.D., n = 3.

Fig. 5.

Loss of LYRM2 leads to respiration defects. A, Mitochondrial proteins were separated by BN-PAGE before NADH-dehydrogenase in-gel activity assay; *, NADH dehydrogenase activity from dihydrolipoamide dehydrogenase (73). B, Oxygen consumption rates of control and LYRM2^KO cell lines using treatments as indicated. Data reported as mean ± S.D. n = 8–9. C, Mitochondria from control, LYRM2^KO, and LYRM2^KO cells stably complemented with Flag LYRM2^Flag were separated by BN-PAGE before Western blotting and incubation with antibodies against the complex I N-module subunit NDUFV1. D, AE-MS was performed on LYRM2^Flag whole cell lysates as in Fig. 1A. Threshold of significance of <1% FDR @ s0 = 1 was used, revealing specifically interacting proteins. Biological replicates n = 3. CI, complex I. CI^ΔN, complex I missing N-module.