MIEF1 Microprotein Regulates Mitochondrial Translation

Abstract

Recent technological advances led to the discovery of hundreds to thousands of peptides and small proteins (microproteins) encoded by small open reading frames (smORFs). Characterization of new microproteins demonstrates their role in fundamental biological processes and highlights the value in discovering and characterizing more microproteins. The elucidation of microprotein-protein interactions (MPIs) is useful for determining the biochemical and cellular roles of microproteins. In this study, we characterize the protein interaction partners of mitochondrial elongation factor 1 microprotein (MIEF1-MP) using a proximity labeling strategy that relies on APEX2. MIEF1-MP localizes to the mitochondrial matrix where it interacts with the mitochondrial ribosome (mitoribosome). Functional studies demonstrate that MIEF1-MP regulates mitochondrial translation via its binding to the mitoribosome. Loss of MIEF1-MP decreases the mitochondrial translation rate, while an elevated level of MIEF1-MP increases the translation rate. The identification of MIEF1-MP reveals a new gene involved in this process.

Figure 1.

MIEF1-MP expression and conservation. (A) MIEF1-MP is encoded by an upstream smORF (uORF) in the MIEF1 mRNA. RNA-Seq (magenta) and Ribo-Seq (green) data from HEK293T cells indicates translation of the MIEF1-MP smORF and the MIEF1 (ORF) . (B) A tryptic peptide from the MIEF1 microprotein detected by shotgun mass spectrometry validates that MIEF1-MP is a stable member of the proteome. (C) Sequence alignment of the MIEF1-MP demonstrates that this microprotein is conserved in numerous species at the protein level.

Figure 2.

MIEF1-MP is a mitochondrial matrix microprotein. (A) MIEF1-MP-FLAG colocalizes with the mitochondrial marker TOM20 in Hela cells verifying its mitochondrial localization (Hoechst (blue) nuclear stain, TOM20 (red), MIEF1-MP (green), yellow/orange overlap). (B) Proteinase K digestion of MIEF1-MP-FLAG had a similar pattern to HSP60, a mitochondrial matrix marker, confirming its sub-cellular localization to the mitochondrial matrix in HEK293T cells.

Figure 3.

MIEF1-MP contains an LYR motif and interacts with ACPM/ NDUFAB1 when overexpressed. (A) A LOGO plot of MIEF1-MP with known LYR motif containing proteins highlights the conserved tripeptide L-Y-R (leucine/tyrosine/arginine) and downstream F (phenylalanine) (LYR and F) motif. (B) Immunoprecipitation (IP) of the MIEF1-MP-FLAG enriches ACPM/NDUFAB1 while mutating the LYR and F. to alanines abrogates this binding. (C) Proximity labeling with MIEF1-MP-APEX-MYC demonstrates ACPM/NDUFAB1 enrichment in living cells.

Figure 4.

MIEF1-MP interacts with the mitoribosome. (A) The interaction network of the proteins enriched in the MIEF1-MP-APEX-MYC experiment after STRING analysis reveals MIEF1-MP enriched proteins that interact with each other. (B) Spectral counts for different members of the mitoribosome after the MIEF1-MP-APEX-MYC experiment. (C) Western blot validation of MIEF1-MP protein-protein interaction with some members of the mitoribosome (MRPL4 and MRPL27) by proximity labeling. The experiment used an MIEF1-MP-APEX-MYC expression construct for the proximity labeling and expression of MIEF1-MP-APEX-MYC was confirmed by western blot using anti-MYC antibodies. (D) An “in silico” reverse IP searching for MIEF1-MP peptides in mitoribosome protein data from the Bioplex database. Bar graph shows the number of spectral counts for MIEF1-MP peptides in the pulldown data for each of the mitoribosome proteins on the x-axis from the Bioplex database. In addition to the mitoribosome proteins enriched in the proximity labelling experiment (grey) additional mitoribosome proteins that were able to enrich MIEF1-MP were identified (blue).

Figure 5.

MIEF1-MP modulates the mitochondrial translation rate. (A) Using the BONCAT method, newly synthesized mtDNA encoded proteins are labelled with IRDye® 800CW dye and visualized on a gel. MIEF1-MP-FLAG overexpression in HEK293T cells increases the amount of newly synthesized mitochondrial proteins versus the control (pcDNA vector). (B) MIEF1 siRNA-treated HEK293T cells led to decreased levels of translation of newly synthesized proteins compared to control siRNA treated cells. (C) Quantitative analysis showed robust change for the newly synthesized mtDNA encoded protein levels on MIEF1 microprotein overexpression and knockdown. Comparison of the MIEF1-MP perturbed sample to the control was used to determine statistical significance. For example, MIEF1-MP overexpression leads to a statistically significant increase in MT-ND4 versus the pcDNA control, and MIEF1-MP siRNA leads to a statistically significant decrease in MT-ND4 translation versus the siRNA control (*, p-value <0.05 using t-test).

Figure 6.. Rescuing the effect of MIEF1 siRNA treatment on mitochondrial translation rate.

Using the BONCAT method, newly synthesized mtDNA encoded proteins are labelled with IRDye® 800CW dye and visualized on a gel. A) In the MIEF1 siRNA treated sample expressing MIEF1-MP-FLAG increases the amount of newly synthesized mitochondrial proteins versus the control (pcDNA vector). B) Quantitative analysis showed robust change for the newly synthesized mtDNA encoded protein levels on MIEF1 microprotein knockdown and re-expression. Comparison of the MIEF1 siRNA treated with MIEF1-MP re-expressed sample to the control was used to determine statistical significance (*, p-value <0.05 using t-test).