Functional analyses of mitoribosome 54S subunit devoid of mitochondria‐specific protein sequences

Abstract

In Saccharomyces cerevisiae, mitoribosomes are composed of a 54S large subunit (mtLSU) and a 37S small subunit (mtSSU). The two subunits altogether contain 73 mitoribosome proteins (MRPs) and two ribosomal RNAs (rRNAs). Although mitoribosomes preserve some similarities with their bacterial counterparts, they have significantly diverged by acquiring new proteins, protein extensions, and new RNA segments, adapting the mitoribosome to the synthesis of highly hydrophobic membrane proteins. In this study, we investigated the functional relevance of mitochondria‐specific protein extensions at the C‐terminus (C) or N‐terminus (N) present in 19 proteins of the mtLSU. The studied mitochondria‐specific extensions consist of long tails and loops extending from globular domains that mainly interact with mitochondria‐specific proteins and 21S rRNA moieties extensions. The expression of variants devoid of extensions in uL4 (C), uL5 (N), uL13 (N), uL13 (C), uL16 (C), bL17 (N), bL17 (C), bL21 (24), uL22 (N), uL23 (N), uL23 (C), uL24 (C), bL27 (C), bL28 (N), bL28 (C), uL29 (N), uL29 (C), uL30 (C), bL31 (C), and bL32 (C) did not rescue the mitochondrial protein synthesis capacities and respiratory growth of the respective null mutants. On the contrary, the truncated form of the mitoribosome exit tunnel protein uL24 (N) yields a partially functional mitoribosome. Also, the removal of mitochondria‐specific sequences from uL1 (N), uL3 (N), uL16 (N), bL9 (N), bL19 (C), uL29 (C), and bL31 (N) did not affect the mitoribosome function and respiratory growth. The collection of mutants described here provides new means to study and evaluate defective assembly modules in the mitoribosome biogenesis process.

Overexpression of MRX9 impairs processing of RNAs encoding mitochondrial oxidative phosphorylation factors COB and COX1 in yeast

“…S2 ). Similar increments in paromomycin resistance were observed before with mitoribosome mutants ( 52 ).…”

“…We also evaluate whether the mrx9 deletion would have an additive effect on mitoribosome mutants. Recently, we described new LSU mitoribosome mutants, some of them with poor growth on respiratory media ( 52 ); considering the proximity of Mrx9p with the LSU tunnel exit components, particularly uL23, mL57, and uL29 ( 41 ), we hypothesized that Mrx9p may play a role in the mitoribosome biogenesis. If this was the case, then its removal should have an additive effect over the slow respiratory growth of uL16, uL23, or uL29 mutants ( 52 ); likewise the aforementioned phenotype of shy1 mrx9 double mutant, mrx9 removal marginally worsens the respiratory growth of the uL23 mutant ( Fig.…”

“…Mitochondrial translation products were labeled in whole cells with a mixture of [ 35 S] methionine and [ 35 S] cysteine (7 mCi/mmol) in the presence of cycloheximide. Cells were lysed and protein extracted as previously described ( 52 ). Total cellular proteins were separated by SDS-PAGE on 17.5% polyacrylamide or 6M UREA-PAGE on 12.5% polyacrylamide gel systems.…”

The N‐terminal region of yeast mitoribosomal Mrp7/bL27m protein serves to optimize translation of nascent chains competent for OXPHOS complex assembly

“…These mitospecific protein elements include both novel proteins unique to mitoribosomes as well as extension sequences (usually C‐terminal extensions) on some of the mitoribosomal proteins conserved from bacterial ribosomes. These mitospecific protein features are thought to contribute to mitochondrial‐specific aspects of translation, for example, the permanent membrane tethering of mitoribosomes and potentially, depending on the metabolic environment of the cell, ensuring that the mitotranslational process is coordinated with cytosolic ribosome activity and the import of nuclear‐encoded proteins [13–16].…”