The Membrane-bound GTPase Guf1 Promotes Mitochondrial Protein Synthesis under Suboptimal Conditions

Abstract

Recently, the bacterial elongation factor LepA was identified as critical for the accuracy of in vitro translation reactions. Extremely well conserved homologues of LepA are present throughout bacteria and eukaryotes, but the physiological relevance of these proteins is unclear. Here we show that the yeast counterpart of LepA, Guf1, is located in the mitochondrial matrix and tightly associated with the inner membrane. It binds to mitochondrial ribosomes in a GTP-dependent manner. Mutants lacking Guf1 show cold-and heat-sensitive growth defects on non-fermentable carbon sources that are especially pronounced under nutrient-limiting conditions. The cold sensitivity is explained by diminished rates of protein synthesis at low temperatures. At elevated temperatures, Guf1-deficient mutants exhibit defects in the assembly of cytochrome oxidase, suggesting that the polypeptides produced are not functional. Moreover, Guf1 mutants exhibit synthetic growth defects with mutations of the protein insertase Oxa1. These observations show a critical role for Guf1 in vivo. The observed defects in Guf1-deficient mitochondria are consistent with a function of Guf1 as a fidelity factor of mitochondrial protein synthesis.

West syndrome caused by homozygous variant in the evolutionary conserved gene encoding the mitochondrial elongation factor GUF1

“…In budding yeasts, GUF1 is important for mitochondrial protein synthesis under suboptimal conditions. 23 Yeasts that lack the GUF1 (Δguf1) elongation factor show a mild growth defect under extreme growth-limiting concentrations of non-fermentable carbon sources and low and high temperature. On the contrary, overexpression of Guf1 exhibits a pronounced negative effect, 23 consistent with the observation that overproduction of LepA stalls translation in bacteria.…”

“…23 Yeasts that lack the GUF1 (Δguf1) elongation factor show a mild growth defect under extreme growth-limiting concentrations of non-fermentable carbon sources and low and high temperature. On the contrary, overexpression of Guf1 exhibits a pronounced negative effect, 23 consistent with the observation that overproduction of LepA stalls translation in bacteria. 30 We found that overexpression of hGUF1 and hGUF1 A609S in Δguf1 had comparable effect on growth, mitochondrial protein expression and translation (Supplementary Figure S5A and D and Supplementary Materials).…”

“…Upon incubation at elevated growth temperatures (37°C), the Δguf1 strains showed a mild growth defect confirming earlier studies. 23 The growth defect was further aggravated by overexpression of either hGUF1 or hGUF1 A609S , indicating that both variants interfere with mitochondrial translation machinery with similar degrees. Next we evaluated whether the expression of the human Guf1 proteins influences mitochondrial protein expression.…”

“…To assess mitochondrial protein synthesis more directly, we incubated mitochondria in the presence of ATP, amino acids and radiolabeled [ 35 S]-methionine for 10 min at either 15 or 30°C. Mitochondria were then reisolated, and newly synthesized translation products were visualized by autoradiography as described in Bauerschmitt et al 23 Again, no differences were observed between Δguf1 and Δguf1-overexpressing hGUF1 or hGUF1 A609S cells, all strains showing a similar translation pattern. Whereas deletion of GUF1 leads to almost no detectable defects in yeast, Guf1 activity is critical in the oxa1ΔC mutant.…”

“…Whereas deletion of GUF1 leads to almost no detectable defects in yeast, Guf1 activity is critical in the oxa1ΔC mutant. 23 This strain lacks part of the C-terminal ribosomebinding domain of the mitochondrial Oxa1 insertase (residues 332-402). It shows no obvious defect in the biogenesis of mitochondrial translation products 24,25 but exhibits a strong synthetic growth defect with Δguf1 at high temperatures and on non-fermentable carbon sources.…”