GTPBP8 is required for mitoribosomal biogenesis and mitochondrial translation

Mitochondrial translation occurs on the mitochondrial ribosome, also known as the mitoribosome. The assembly of mitoribosomes is a highly coordinated process. During mitoribosome biogenesis, various assembly factors transiently associate with the nascent ribosome, facilitating the accurate and efficient construction of the mitoribosome. However, the specific factors involved in the assembly process, the precise mechanisms, and the cellular compartments involved in this vital process are not yet fully understood. In this study, we discovered a crucial role for GTP-binding protein 8 (GTPBP8) in the assembly of the mitoribosomal large subunit (mt-LSU) and mitochondrial translation. GTPBP8 is identified as a novel GTPase located in the matrix and peripherally bound to the inner mitochondrial membrane. Importantly, GTPBP8 is specifically associated with the mt-LSU during its assembly. Depletion of GTPBP8 leads to an abnormal accumulation of mt-LSU, indicating that GTPBP8 is critical for proper mt-LSU assembly. Furthermore, the absence of GTPBP8 results in reduced levels of fully assembled 55S monosomes. This impaired assembly leads to compromised mitochondrial translation and, consequently, impaired mitochondrial function. The identification of GTPBP8 as an important player in these processes provides new insights into the molecular mechanisms underlying mitochondrial protein synthesis and its regulation. Supplementary Information The online version contains supplementary material available at 10.1007/s00018-023-05014-0.


Figure S1
. GTPBP8 is an evolutionarily conserved GTPase.(A) Phylogenetic analysis of GTPBP8.Phylogenetic tree was built using Geneious 6.1.8.(B) Multiple sequence alignment of GTPBP8 orthologue in prokaryotes (P38424 for B. subtilis Ysxc and P0A6P7 for E.coli Yiha) and eukaryotes (Q8N3Z3 for human GTPBP8) using clustal omega server.The GTPase domains (G1-G5) were indicated in red boxes.The N-terminal mitochondrial targeting sequence (1-46aa) was indicated by a magenta bar.(C) The intrinsic GTPase activity of GTPBP8.Data is an average of three independent measurements with error bars (± SD) indicated.

Figure S2. GTPBP8 is a mitochondrial protein. (A)
Immunoblotting detection of the expressed GTPBP8-myc in U2OS cells using an anti-myc antibody.(B) Immunoblotting detection of the expressed GTPBP8-GFP in U2OS cells using an anti-GFP antibody.Cells expressed only the GFPvector were used as a negative control.(C) Immunoblotting analysis of subcellular localization of GTPBP8-myc.After transiently expressing GTPBP8-myc in U2OS cells for 48 h, cells were fractionated and the expressed GTPBP8-myc was detected in the whole-cell lysate (WCL), cytoplasm (CYT) and isolated mitochondria (MIT) with anti-myc and anti-GTPBP8 antibodies.Antibodies against mitochondrial proteins TOM40 and Cytochrome C, and GAPDH were used as controls.(D) Immunoblotting analysis of submitochondrial localization of GTPBP8-myc using proteinase K digestion of isolated mitochondria from GTPBP8-myc expressed cells.Mitochondria were subjected to hypotonic swelling or permeabilization with 1% NP-40, treated with 100 μg/mL proteinase K as indicated.

Figure S3
. Effects of GTPBP8 depletion on protein abundance of the oxidative phosphorylation complexes and functional networks by mass spectroscopy analysis.(A) Main functionally affected biological processes by GTPBP8 depletion.(B) Steady-state protein levels of oxidative phosphorylation complexes after depletion of GTPBP8.

Figure S5 .
Figure S5.Loss of GTPBP8 caused defects in mitochondrial translation.(A) Effects of GTPBP8 loss on the steady state levels of mitoribosomal proteins.GTPBP8 was depleted using siRNA #2.(B) Metabolic labeling of mitochondrial translation products with [ 35 S]-methionine/cysteine in U2OS cells treated with GTPBP8 siRNA #2 or expression of GTPBP8-myc.(C) Quantification of 35 S intensity in panel B for all the mtDNA-encoded translation products relative to the corresponding intensity in coomassie staining.

Figure S6 .
Figure S6.The steady state levels of GTPBP8.Immunoblotting for the steady state levels of GTPBP8 in the wild type, GTPBP8-depleted, and rescue cell samples.