Human GTPBP5 is involved in the late stage of mitoribosome large subunit assembly

Abstract

Human mitoribosomes are macromolecular complexes essential for translation of 11 mitochondrial mRNAs. The large and the small mitoribosomal subunits undergo a multistep maturation process that requires the involvement of several factors. Among these factors, GTP-binding proteins (GTPBPs) play an important role as GTP hydrolysis can provide energy throughout the assembly stages. In bacteria, many GTPBPs are needed for the maturation of ribosome subunits and, of particular interest for this study, ObgE has been shown to assist in the 50S subunit assembly. Here, we characterize the role of a related human Obg-family member, GTPBP5. We show that GTPBP5 interacts specifically with the large mitoribosomal subunit (mt-LSU) proteins and several late-stage mitoribosome assembly factors, including MTERF4:NSUN4 complex, MRM2 methyltransferase, MALSU1 and MTG1. Interestingly, we find that interaction of GTPBP5 with the mt-LSU is compromised in the presence of a non-hydrolysable analogue of GTP, implying a different mechanism of action of this protein in contrast to that of other Obg-family GTPBPs. GTPBP5 ablation leads to severe impairment in the oxidative phosphorylation system, concurrent with a decrease in mitochondrial translation and reduced monosome formation. Overall, our data indicate an important role of GTPBP5 in mitochondrial function and suggest its involvement in the late-stage of mt-LSU maturation.

Structural basis for late maturation steps of the human mitoribosomal large subunit

“…4b). These structural conclusions are supported by earlier mass-spectrometry data on isolated GTPBP5 IP assembly intermediates and protein-proximity interactome analysis 8,28 .…”

“…Multiple assembly factors, many specific to mammalian mitochondria, assist in the mitoribosome assembly process. A growing number of studies in recent years have shown that a family of GTP-binding proteins (GTPBPs) is crucial for mammalian mitoribosome assembly [8][9][10][11][12][13] . Among these GTPBPs, GTPBP5 participates in the late steps of large subunit (mt-LSU) maturation, and its deletion leads to severe translational defects 8,11 .…”

“…6a,c). The requirement for GTPBP5 to be in a GTP-bound state is supported by the inability of a GTPBP5 Walker A mutant (GTPBP5-S238A) to bind mt-LSU intermediates 8 . A back-rotation of the L7/12 stalk, presumably by binding of another maturation factor to the mt-LSU assembly intermediate, would lead to a release of the switch I and the activation of GTP hydrolysis, followed by release of GTPBP5 from the mt-LSU assembly intermediate.…”

“…The knock-out cell line (GTPBP5 KO ) was generated in the Flp-In T-Rex human embryonic kidney 293 (HEK293T) cell line (Invitrogen) using CRISPR/Cas9 technology targeted on exon 1 of MTG2 gene, which encodes for GTPBP5, as described 8 . In short, two pairs of gRNAs were designed and cloned into the pSpCas9(BB)-2A-Puro (pX459) V2.0 vector to generate outof-frame deletions.…”

“…A stable mammalian cell line overexpressing C-terminal FLAG-tagged GTPBP5 (GTPBP5::FLAG) in a doxycycline-inducible dose-dependent manner was generated as described in Cipullo et al 8 Penicillin/Streptomycin (Gibco), 50 µmg/ml uridine, 10 µg/ml Zeocin (Invitrogen) and 100 µg/ml blasticidin (Gibco) at 37 °C under 5% CO2 atmosphere. Cells were seeded in a 6-well plate, grown in medium without antibiotics and co-transfected with pcDNA5/FRT/TO-GTPBP5::FLAG and pOG44 using Lipofectamine 3000 according to manufacturer's recommendations.…”