Importance of conserved hydrophobic pocket region in yeast mitoribosomal mL44 protein for mitotranslation and transcript preference.

The mitochondrial ribosome (mitoribosome) is responsible for the synthesis of key oxidative phosphorylation subunits encoded by the mitochondrial genome. Defects in mitoribosomal function therefore can have serious consequences for the bioenergetic capacity of the cell. Mutation of the conserved mitoribosomal mL44 protein has been directly linked to childhood cardiomyopathy and progressive neurophysiology issues. To further explore the functional significance of the mL44 protein in supporting mitochondrial protein synthesis we have performed a mutagenesis study of the yeast mL44 homolog, the MrpL3/mL44 protein. We specifically investigated the conserved hydrophobic pocket region of the MrpL3/mL44 protein, where the known disease-related residue in the human mL44 protein (L156R) is located. While our findings identify a number of residues in this region critical for MrpL3/mL44's ability to support the assembly of translationally active mitoribosomes, the introduction of the disease-related mutation into the equivalent position in the yeast protein (residue A186) was found not have a major impact on function. The human and yeast mL44 proteins share many similarities in sequence and structure, however results presented here indicate that these two proteins have diverged somewhat in evolution. Finally, we observed that mutation of the MrpL3/mL44 does not impact the translation of all mitochondrial encoded proteins equally, suggesting the mitochondrial translation system may exhibit a transcript hierarchy and prioritization.

Mutation of the PEBP-like domain of the mitoribosomal MrpL35/mL38 protein results in production of nascent chains with impaired capacity to assemble into OXPHOS complexes.

Located in the central protuberance region of the mitoribosome and mitospecific mL38 proteins display homology to PEBP (Phosphatidylethanolamine Binding Protein) proteins, a diverse family of proteins reported to bind anionic substrates/ligands and implicated in cellular signaling and differentiation pathways. In this study, we have performed a mutational analysis of the yeast mitoribosomal protein MrpL35/mL38 and demonstrate that mutation of the PEBP-invariant ligand binding residues Asp(D)232 and Arg(R)288 impacted MrpL35/mL38's ability to support OXPHOS-based growth of the cell. Furthermore, our data indicate these residues exist in a functionally important charged microenvironment, which also includes Asp(D)167 of MrpL35/mL38 and Arg(R)127 of the neighboring Mrp7/bL27m protein. We report that mutation of each of these charged residues resulted in a strong reduction in OXPHOS complex levels that was not attributed to a corresponding inhibition of the mitochondrial translation process. Rather, our findings indicate that a disconnect exists in these mutants between the processes of mitochondrial protein translation and the events required to ensure the competency and/or availability of the newly synthesized proteins to assemble into OXPHOS enzymes. Based on our findings, we postulate that the PEBP-homology domain of MrpL35/mL38, together with its partner Mrp7/bL27m, form a key regulatory region of the mitoribosome.

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

The extreme N-terminal residues of the mitochondrial ribosomal bL27m proteins reside within the ribosomal peptidyl transferase center (PTC) and are conserved from their bacterial ancestors. Mutation or truncation of the N-terminal region of the yeast Mrp7/bL27m protein did not inhibit protein synthesis but significantly impacted the efficacy of the mitochondrial translational process with respect to yielding proteins competent to assemble into functional oxidative phosphorylation enzymes. The requirement for the N-terminal residues of Mrp7/bL27m to support normal mitotranslation was more apparent under respiratory growth. We demonstrate that the N-terminal region of Mrp7/bL27m impacts the environment of the PTC and speculate the bL27m proteins serve to fine-tune and optimize mitoribosomal activity with respect to the downstream fate of the nascent chain.

The mitospecific domain of Mrp7 (bL27) supports mitochondrial translation during fermentation and is required for effective adaptation to respiration.

We demonstrate here that mitoribosomal protein synthesis, responsible for the synthesis of oxidative phosphorylation (OXPHOS) subunits encoded by the mitochondrial genome, occurs at high levels during glycolysis fermentation and in a manner uncoupled from OXPHOS complex assembly regulation. Furthermore, we provide evidence that the mitospecific domain of Mrp7 (bL27), a mitoribosomal component, is required to maintain mitochondrial protein synthesis during fermentation but is not required under respiration growth conditions. Maintaining mitotranslation under high-glucose-fermentation conditions also involves Mam33 (p32/gC1qR homologue), a binding partner of Mrp7's mitospecific domain, and together they confer a competitive advantage for a cell's ability to adapt to respiration-based metabolism when glucose becomes limiting. Furthermore, our findings support that the mitoribosome, and specifically the central protuberance region, may be differentially regulated and/or assembled, under the different metabolic conditions of fermentation and respiration. On the basis of our findings, we propose that the purpose of mitotranslation is not limited to the assembly of OXPHOS complexes, but also plays a role in mitochondrial signaling critical for switching cellular metabolism from a glycolysis- to a respiration-based state.

MrpL35, a mitospecific component of mitoribosomes, plays a key role in cytochrome c oxidase assembly.

Mitoribosomes perform the synthesis of the core components of the oxidative phosphorylation (OXPHOS) system encoded by the mitochondrial genome. We provide evidence that MrpL35 (mL38), a mitospecific component of the yeast mitoribosomal central protuberance, assembles into a subcomplex with MrpL7 (uL5), Mrp7 (bL27), and MrpL36 (bL31) and mitospecific proteins MrpL17 (mL46) and MrpL28 (mL40). We isolated respiratory defective mrpL35 mutant yeast strains, which do not display an overall inhibition in mitochondrial protein synthesis but rather have a problem in cytochrome c oxidase complex (COX) assembly. Our findings indicate that MrpL35, with its partner Mrp7, play a key role in coordinating the synthesis of the Cox1 subunit with its assembly into the COX enzyme and in a manner that involves the Cox14 and Coa3 proteins. We propose that MrpL35 and Mrp7 are regulatory subunits of the mitoribosome acting to coordinate protein synthesis and OXPHOS assembly events and thus the bioenergetic capacity of the mitochondria.

Genetic basis for Rhizobium etli CE3 O-antigen O-methylated residues that vary according to growth conditions.

The Rhizobium etli CE3 O antigen is a fixed-length heteropolymer with O methylation being the predominant type of sugar modification. There are two O-methylated residues that occur, on average, once per complete O antigen: a multiply O-methylated terminal fucose and 2-O methylation of a fucose residue within a repeating unit. The amount of the methylated terminal fucose decreases and the amount of 2-O-methylfucose increases when bacteria are grown in the presence of the host plant, Phaseolus vulgaris, or its seed exudates. Insertion mutagenesis was used to identify open reading frames required for the presence of these O-methylated residues. The presence of the methylated terminal fucose required genes wreA, wreB, wreC, wreD, and wreF, whereas 2-O methylation of internal fucoses required the methyltransferase domain of bifunctional gene wreM. Mutants lacking only the methylated terminal fucose, lacking only 2-O methylation, or lacking both the methylated terminal fucose and 2-O methylation exhibited no other lipopolysaccharide structural defects. Thus, neither of these decorations is required for normal O-antigen length, transport, or assembly into the final lipopolysaccharide. This is in contrast to certain enteric bacteria in which the absence of a terminal decoration severely affects O-antigen length and transport. R. etli mutants lacking only the methylated terminal fucose were not altered in symbiosis with host Phaseolus vulgaris, whereas mutants lacking only 2-O-methylfucose exhibited a delay in nodule development during symbiosis. These results support previous conclusions that the methylated terminal fucose is dispensable for symbiosis, whereas 2-O methylation of internal fucoses somehow facilitates early events in symbiosis.

2-O-methylation of fucosyl residues of a rhizobial lipopolysaccharide is increased in response to host exudate and is eliminated in a symbiotically defective mutant.

When Rhizobium etli CE3 was grown in the presence of Phaseolus vulgaris seed extracts containing anthocyanins, its lipopolysaccharide (LPS) sugar composition was changed in two ways: greatly decreased content of what is normally the terminal residue of the LPS, di-O-methylfucose, and a doubling of the 2-O-methylation of other fucose residues in the LPS O antigen. R. etli strain CE395 was isolated after Tn5 mutagenesis of strain CE3 by screening for mutant colonies that did not change antigenically in the presence of seed extract. The LPS of this strain completely lacked 2-O-methylfucose, regardless of whether anthocyanins were present during growth. The mutant gave only pseudonodules in association with P. vulgaris. Interpretation of this phenotype was complicated by a second LPS defect exhibited by the mutant: its LPS population had only about 50% of the normal amount of O-antigen-containing LPS (LPS I). The latter defect could be suppressed genetically such that the resulting strain (CE395 alpha 395) synthesized the normal amount of an LPS I that still lacked 2-O-methylfucose residues. Strain CE395 alpha 395 did not elicit pseudonodules but resulted in significantly slower nodule development, fewer nodules, and less nitrogenase activity than lps(+) strains. The relative symbiotic deficiency was more severe when seeds were planted and inoculated with bacteria before they germinated. These results support previous conclusions that the relative amount of LPS I on the bacterial surface is crucial in symbiosis, but LPS structural features, such as 2-O-methylation of fucose, also may facilitate symbiotic interactions.