Phosphorylated proteins of the mammalian mitochondrial ribosome: implications in protein synthesis

Abstract

Mitochondria, the powerhouse of eukaryotic cells, have their own translation machinery that is solely responsible for synthesis of 13 mitochondrially encoded protein subunits of oxidative phosphorylation complexes. Phosphorylation is a well-known post-translational modification in regulation of many processes in mammalian mitochondria including oxidative phosphorylation. However, there is still very limited knowledge on phosphorylation of mitochondrial ribosomal proteins and their role(s) in ribosome function. In this study, we have identified the mitochondrial ribosomal proteins that are phosphorylated at serine, threonine or tyrosine residues. Twenty-four phosphorylated proteins were visualized by phosphorylation-specific techniques including in vitro radiolabeling, residue specific antibodies for phosphorylated residues, or ProQ phospho dye and identified by tandem mass spectrometry. Translation assays with isolated ribosomes that were phosphorylated in vitro by kinases PKA, PKCdelta, or Abl Tyr showed up to 30% inhibition due to phosphorylation. Findings from this study should serve as the framework for future studies addressing the regulation mechanisms of mitochondrial translation machinery by phosphorylation and other post-translational modifications.

Figure 1

Two-dimensional gel analysis of in vitro phosphorylated mitochondrial ribosomal proteins using [γ-³²P] ATP. Bovine mitochondrial ribosomes (1.8 A₂₆₀ units) were incubated in the presence of 50 μCi [γ-³²P] ATP for 1 hr at 30 °C and separated on non-equilibrium pH gradient electrophoresis (NEPHGE) gels using pI 3-10 and 8-10 ampholytes. The phosphorylated proteins were excised, digested with trypsin, and analyzed by LC-MS/MS for potential phosphorylation sites using an ion trap mass spectrometer. (A) Coomassie Blue stained gel and (B) Phosphor-image of mitochondrial ribosomal proteins obtained from purified 55S ribosomes. Not all of the phosphorylated proteins identified by mass spectrometry are labeled in these 2D-gels.

Figure 2

Mitochondrial ribosomes were phosphorylated in the presence of endogenous and mitochondrially located kinases to determine the effect on mitochondrial translation. Approximately, 0.4 A₂₆₀ units of mitochondrial ribosomes were incubated with 5 μCi [γ-³²P] ATP and 2500 U of cAMP-dependent protein kinase (PKA) the catalytic subunit, 48 ng of protein kinase C delta (PKCδ), and 100 U of Abl protein Tyr kinase at 30 °C for 1 hr. The ribosome in the presence of the kinase buffer served as the control. (A) The ribosomal samples were run on an SDS-PAGE gel, fixed, dried, and visualized by phosphor-imaging. The upper arrow indicates the autophosphorylation of PKCδ and lower arrow is highlighting the phosphorylation of pyruvate dehydrogenase subunit. (B) After phosphorylating the ribosome with different kinases, poly (U)-directed polymerization assays were conducted. Shown is the mean ± SD of three independent experiments. *, P < 0.05.

Figure 3

Two-dimensional gel analysis of phosphorylated mitochondrial ribosomal proteins. Approximately, 1.8 A₂₆₀ units of bovine mitochondrial ribosomes were separated on non-equilibrium pH gradient electrophoresis (NEPHGE) gels using pI 3-10 and 8-10 ampholytes. The phosphorylated proteins were excised, digested with trypsin, and analyzed by LC-MS/MS for potential phosphorylation sites using an ion trap mass spectrometer. (A) SYPRO Ruby stained gel of the ribosomal proteins obtained from bovine 55S ribosomes. (B and C) Immunoblots of 55S ribosomes using anti-phosphoserine and anti-phosphotyrosine antibodies, respectively. Not all of the phosphorylated proteins identified by mass spectrometry are labeled in these 2D-gels for simplicity. (D) ProQ Diamond stained gel of the phosphorylated proteins from 55S ribosomes visualized at 532 nm.

Figure 4

3D-Models of the E. coli ribosomal subunits displaying the location of phosphorylated mitochondrial ribosomal proteins. The phosphorylated ribosomal proteins are highlighted in different colors, while gray represents unphosphorylated ribosomal proteins, and blue is the rRNA. Coordinates of the E. coli 30S subunit and 50S subunit were obtained from the Protein Data Bank (Acc. # 2AW7 and 2AW4). (A) The 30S subunit is illustrated from the solvent side, while (B) represents a view of the small subunit from the 50S interface. (C) Representation of the 50S subunit from the 30S interface, while (D) is a view of the large subunit from the solvent side. Specific regions, peptidyl transferase center (PTC), central protuberance (CP), sarcin-ricin loop (SRL), L1 and L7/L12 stalks, exit tunnel, and the phosphorylated ribosomal proteins were labeled in the model generated by PyMOL software.

Figure 4

3D-Models of the E. coli ribosomal subunits displaying the location of phosphorylated mitochondrial ribosomal proteins. The phosphorylated ribosomal proteins are highlighted in different colors, while gray represents unphosphorylated ribosomal proteins, and blue is the rRNA. Coordinates of the E. coli 30S subunit and 50S subunit were obtained from the Protein Data Bank (Acc. # 2AW7 and 2AW4). (A) The 30S subunit is illustrated from the solvent side, while (B) represents a view of the small subunit from the 50S interface. (C) Representation of the 50S subunit from the 30S interface, while (D) is a view of the large subunit from the solvent side. Specific regions, peptidyl transferase center (PTC), central protuberance (CP), sarcin-ricin loop (SRL), L1 and L7/L12 stalks, exit tunnel, and the phosphorylated ribosomal proteins were labeled in the model generated by PyMOL software.

Figure 5

Mapping of phosphorylation site(s) in mitochondrial ribosomal proteins by MS/MS. (A) A tryptic peptide (TANAEAVVYGHGSGK, m/z 730.91, 2+) obtained from MS/MS analysis of MRPS9 spot was presented. (B) The CID spectrum of the phosphorylated form of the same peptide pTANAEAVVYGHGSGK (m/z 770.93, 2+) at the first Thr residue was shown (C) The phosphopeptide obtained from bovine MRPS9 was aligned with the same region of MRPS9 homologs from dog (XP_531774), bovine (Q58DQ5), human (P82933), mouse (Q9D7N3), chicken (XP_416921), thernus (P80374), and E. coli (P0A7X3) using CLUSTALW program in Biology Workbench and the results are displayed in BOXSHADE. The asterisk indicates the site of phosphorylation mapped for MRPS9 peptide.

Figure 5

Mapping of phosphorylation site(s) in mitochondrial ribosomal proteins by MS/MS. (A) A tryptic peptide (TANAEAVVYGHGSGK, m/z 730.91, 2+) obtained from MS/MS analysis of MRPS9 spot was presented. (B) The CID spectrum of the phosphorylated form of the same peptide pTANAEAVVYGHGSGK (m/z 770.93, 2+) at the first Thr residue was shown (C) The phosphopeptide obtained from bovine MRPS9 was aligned with the same region of MRPS9 homologs from dog (XP_531774), bovine (Q58DQ5), human (P82933), mouse (Q9D7N3), chicken (XP_416921), thernus (P80374), and E. coli (P0A7X3) using CLUSTALW program in Biology Workbench and the results are displayed in BOXSHADE. The asterisk indicates the site of phosphorylation mapped for MRPS9 peptide.

Figure 5

Mapping of phosphorylation site(s) in mitochondrial ribosomal proteins by MS/MS. (A) A tryptic peptide (TANAEAVVYGHGSGK, m/z 730.91, 2+) obtained from MS/MS analysis of MRPS9 spot was presented. (B) The CID spectrum of the phosphorylated form of the same peptide pTANAEAVVYGHGSGK (m/z 770.93, 2+) at the first Thr residue was shown (C) The phosphopeptide obtained from bovine MRPS9 was aligned with the same region of MRPS9 homologs from dog (XP_531774), bovine (Q58DQ5), human (P82933), mouse (Q9D7N3), chicken (XP_416921), thernus (P80374), and E. coli (P0A7X3) using CLUSTALW program in Biology Workbench and the results are displayed in BOXSHADE. The asterisk indicates the site of phosphorylation mapped for MRPS9 peptide.