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Identification and Characterization of CHCHD1, AURKAIP1, and CRIF1 as New Members of the Mammalian Mitochondrial Ribosome

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Identification and Characterization of CHCHD1, AURKAIP1, and CRIF1 as New Members of the Mammalian Mitochondrial Ribosome

Emine C Koc et al. Front Physiol.

Abstract

Defects in mitochondrial ribosomal proteins (MRPs) cause various diseases in humans. Because of the essential role of MRPs in synthesizing the essential subunits of oxidative phosphorylation (OXPHOS) complexes, identifying all of the protein components involved in the mitochondrial translational machinery is critical. Initially, we identified 79 MRPs; however, identifying MRPs with no clear homologs in bacteria and yeast mitochondria was challenging, due to limited availability of expressed sequence tags (ESTs) in the databases available at that time. With the improvement in genome sequencing and increased sensitivity of mass spectrometry (MS)-based technologies, we have established four previously known proteins as MRPs and have confirmed the identification of ICT1 (MRP58) as a ribosomal protein. The newly identified MRPs are MRPS37 (Coiled-coil-helix-coiled-coil-helix domain containing protein 1-CHCHD1), MRPS38 (Aurora kinase A interacting protein1, AURKAIP1), MRPS39 (Pentatricopeptide repeat-containing protein 3, PTCD3), in the small subunit and MRPL59 (CR-6 interacting factor 1, CRIF1) in the large subunit. Furthermore, we have demonstrated the essential roles of CHCHD1, AURKAIP1, and CRIF1in mitochondrial protein synthesis by siRNA knock-down studies, which had significant effects on the expression of mitochondrially encoded proteins.

Keywords: 55S; MRPL58 (ICT1); MRPL59 (CRIF1); MRPS37 (CHCHD1); MRPS38 (AURKAIP1); MRPS39 (PTCD3); mitochondrial ribosomal proteins; mitochondrial translation.

Figures

Figure 1
Figure 1
Experimental scheme for the purification of mitochondrial ribosomes and 28S and 39S subunits. To obtain purified 55S ribosomes and 28S and 39S subunits using sequential linear sucrose gradients, bovine mitochondrial lysates were prepared at different ionic and detergent conditions to remove mitochondrial metabolic enzymes co-sedimenting with the mitochondrial ribosome. Experimental details are included in Materials and Methods.
Figure 2
Figure 2
Identification and detection of mitochondrial ribosomal proteins and their subunit association by capLC-MS/MS and immunoblotting analyses. (A) Purified mitochondrial (55S) ribosomes (~10 A260) prepared in high salt conditions were dissociated into subunits by sedimentation through a second 10–30% linear sucrose gradient in the presence of 2 mM Mg2+ as described in Figure 1. After the sedimentation of the purified subunits, the same A260 units of 28S and 39S subunits and 55S ribosomes (~0.5) were separated on SDS-PAGE. The gel was cut into 30 pieces for in-gel digestion of protein bands with trypsin and the peptides were analyzed by capLC-MS/MS analysis to identify the proteins present. Gel fractions containing the peptides detected from newly identified MRPs are marked on the image. Proteins identified in these gel pieces are given in Tables S1, S2. (B) Purified 28S and 39S subunits (~0.2 A260) were obtained from the dissociation of purified 55S ribosomes as described in Materials and Methods at 2 mM Mg2+. Proteins present in these preparations were separated on 12% SDS-PAGE and analyzed by immunoblotting using CHCHD1, AURKAIP1, CRIF1, MRPS29, and MRPL47 antibodies to confirm subunit distribution of new MRPs. (C) To confirm the ribosome association of newly identified proteins, crude mitochondrial ribosomes prepared at high salt and detergent conditions were separated on a 10–30% linear sucrose gradient in the presence of 20 mM Mg2+ as described in Figure 1. Distribution of new proteins, CHCHD1, AURKAIP1, and CRIF1, were detected by immunoblotting analyses of sucrose gradient fractions containing dissociated ribosomal subunits (28S and 39S) and 55S ribosomes. Antibodies against two 28S subunit proteins, MRPS18B and MRPS29, and a 39S protein, MRPL47, were used to indicate the locations of the subunits and the 55S ribosome.
Figure 3
Figure 3
Sedimentation of new MRPs with large complexes in human cell lines and mitochondria. (A) Whole cell lysate (WCL) and (B) mitochondrial lysate (MITO) obtained from human cell lines were layered on sucrose cushion preparations. After 16 h centrifugation, post-ribosomal supernatant layers (Layers 1 to 6) and the crude ribosome pellet (P) were collected and analyzed by immunoblotting probed with CHCHD1, AURKAIP1, CRIF1, MRPS29, MRPL47, and HSP60 antibodies. (C) To confirm the RNA-dependent association of new MRPs with mitochondrial ribosomes, the same amount of crude ribosomes, both Control and RNase A-treated, were sedimented on 10–30% sucrose gradients. Equal volumes of sucrose gradient fractions were separated on 12% SDS-PAGE. Sedimentation of new MRPs with ribosomal subunits and 55S ribosomes in the absence and presence of RNase A was analyzed by immunoblotting using CHCHD1, AURKAIP1, CRIF1, MRPS29, and MRPL47 antibodies. HSP60 immunoblotting and the Coomassie Blue stained gel are shown as controls for multimeric mitochondrial complexes sedimenting with the 55S ribosome in control and RNase A-treated gradients.
Figure 4
Figure 4
Role of CHCHD1, AURKAIP1, and CRIF1 knock-down on mitochondrial protein synthesis. (A) Transcript levels of CHCHD1, AURKAIP1, and CRIF1 were shown as an indication of knock-down efficiency in cell lines transfected with control siRNA and siRNAs for corresponding new MRPs. Mitochondrial encoded ND6, COI, COII, ATP6, and 12S rRNA, and nuclear encoded MRPS29 and MRPL47 were examined by RT-PCR reactions in the same samples. RT-PCR reactions with GAPDH were performed as a positive control and relative quantitation of signal intensities was calculated based on GAPDH mRNA levels (or siRNA control cell line) in each knock-down cell line. (B) Expression levels of new MRPs in control siRNA and MRP-specific siRNA transfected HEK293T cell lysates and total crude ribosome fractions (*) were analyzed by immunoblotting using antibodies against AURKAIP1, CHCHD1, CRIF1, MRPS29, MRPL47, and HSP60. Immunoblotting analyses with HSP60, MRPS29, and MRPL47 antibodies were used as loading controls for total protein and total ribosome content using whole cell lysates obtained from control and AURKAIP1 siRNA knock-down cells. Similar results were obtained with immunoblotting analyses of total crude ribosome fractions from CHCHD1 and CRIF1 siRNA knock-down cell lines using HSP60, MRPS29, and MRPL47 antibodies (data not shown). (C) De novo synthesis of mitochondrial proteins was evaluated in control siRNA and the siRNA of corresponding MRPs transfected HEK293T cells by pulse labeling of proteins in the presence of [35S]-methionine and a cytosolic translation inhibitor, emetine. A representative electrophoretic pattern of the de novo synthesized translational products is presented. ND1, −2, −3, −4, −4L, −5, and −6 are subunits of Complex I; Cytb is a subunit of Complex III; COI, −II, and −III are subunits of Complex IV; ATP6 and ATP8 are subunits of Complex V. Coomassie blue staining of the same gel was performed to ensure equal protein loading in the gel. (D) The combined intensities of the 13 mitochondrially-encoded proteins from each lane were used as an overall quantitation of mitochondrial protein synthesis from three separate experiments. (E) Immunoblotting analysis of whole cell lysates prepared from control siRNA and MRP specific siRNA transfected HEK293T cells was performed to examine the steady state levels of the mitochondrial encoded subunit of Complex IV (COII) and nuclear encoded subunits of Complex V (ATP5A), III (UQCRC2), and II (SDHB). The arrow shows the reduced translation of mitochondrial encoded protein COII in cell lines transfected with CHCHD1, AURKAIP1, and CRIF1 specific siRNAs. The same PVDF was reprobed with MRPS29, MRPL47, and HSP60 antibodies to ensure equal loading.

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