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.