Kinetics and Mechanism of Mammalian Mitochondrial Ribosome Assembly

Abstract

Summary Mammalian mtDNA encodes only 13 proteins, all essential components of respiratory complexes, synthesized by mitochondrial ribosomes. Mitoribosomes contain greatly truncated RNAs transcribed from mtDNA, including a structural tRNA in place of 5S RNA as a scaffold for binding 82 nucleus-encoded proteins, mitoribosomal proteins (MRPs). Cryoelectron microscopy (cryo-EM) studies have determined the structure of the mitoribosome, but its mechanism of assembly is unknown. Our SILAC pulse-labeling experiments determine the rates of mitochondrial import of MRPs and their assembly into intact mitoribosomes, providing a basis for distinguishing MRPs that bind at early and late stages in mitoribosome assembly to generate a working model for mitoribosome assembly. Mitoribosome assembly is a slow process initiated at the mtDNA nucleoid driven by excess synthesis of individual MRPs. MRPs that are tightly associated in the structure frequently join the complex in a coordinated manner. Clinically significant MRP mutations reported to date affect proteins that bind early on during assembly.

Selective over-synthesis and rapid turnover of mitochondrial protein components of respiratory complexes

“…We selected 19 abundant proteins that were not part of large multi-protein complexes as internal "standards" that showed similar labeling kinetics ( Fig 1A). In general the rate of accumulation of newly synthesized protein slightly exceeds that predicted by our mathematical model (dashed blue line in Fig 1A), which calculates expected H:L ratios based on the cell replication rate assuming that pre-existing light proteins are relatively stable during our experiments as described (2). This heuristic assumption is, of course, incorrect due to protein turnover, which is a variable but minor factor for most proteins in short term labeling experiments.…”

“…We find remarkable differences in the rates at which individual components of respiratory complexes are synthesized and imported into mitochondria. This is not unexpected since we recently reported that assembly of the mitochondrial ribosomes involves a degree of oversynthesis of some of the 80 nucleus-encoded proteins (2). We reasoned that this excess synthesis might be useful to provide an adequate supply of parts for the assembly process.…”

“…The proteins were fragmented with trypsin and peptides were analyzed by LC-MS/MS to determine the relative quantity of labeled (H) and unlabeled (L) peptides. The biochemical preparations and data handling procedures used to generate customized reports of protein H:L ratios were as described (2). Table S1 reports labeling data on several hundred proteins categorized as mitochondrial proteins in the Mitocarta database (6), although the coverage (number of peptides observed) of many less-abundant proteins is often low or inconsistent.…”

“…Labeling with stable isotopes enables mass spectrometric detection of the heavy:light or H:L ratio of a large fraction of the mitochondrial proteome as an index of the synthesis rate for a far greater number of proteins than in a radioactive labeling experiment. When labeling is done on non-synchronized exponentiallygrowing cells, we can make the steady-state assumption that total cell protein should double during each cell generation and calculate the expected H:L ratio at various labeling times (2). We found that some RC, such as F1/Fo ATPase (Complex V) are assembled efficiently with little subunit wastage, while others include some subunits that are imported in considerable excess, as reflected by elevated H:L ratios.…”

“…First, we showed that a subset of newly-synthesized mitoribosomal proteins (MRPs) associated selectively with the mtDNA nucleoid, consistent with their binding to nascent rRNA transcripts (1). We then used SILAC in a pulse-chase labeling format to generate a coarse-grained model for assembly of the mitoribosome (2). One of the principles of this approach is that when a multi-subunit structure is analyzed after pulse-labeling the last proteins added to complete its construction have the highest content of pulse label.…”