2018 Feb 13
Kinetics and Mechanism of Mammalian Mitochondrial Ribosome Assembly
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Kinetics and Mechanism of Mammalian Mitochondrial Ribosome Assembly
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.
mitochondria; mitochondrial biogenesis; proteomics; ribosome assembly.
Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.
Figure 1. SILAC Labeling Resolves Steps in Mitoribosome Assembly
(A) The SILAC pulse-labeling approach. (B) Newly synthesized rRNA is shown as a hypothetical folded structure emanating from mtRNA polymerase (R) with 12S rRNA (orange) still linked to tRNA
V and 16S rRNA (red) as in the RNA 4 precursor. The actual conformation of the nascent RNA is unknown. MRP polypeptides begin to bind nascent rRNA at the nucleoid (Bogenhagen et al., 2014) and assembly intermediates progress to complete mitoribosomes after some delay. In contrast, latebinding proteins are less likely to be found in nucleoid preparations and appear more rapidly in fully assembled, mature mitoribosomes at rates distinguishable by SILAC pulse labeling.
Figure 2. MRPs Appear in Mitoribosomes at Different Rates
(A and B) Scatterplots of H:L ratios observed for individual LSU (A) and SSU (B) MRPs after 3 and 4 hours of SILAC labeling. Points representing MRPs that have consistently low H:L ratios (Table S1) are designated class A (red), while those that appear more rapidly in mitoribosomes are class C (blue). For the larger set of LSU polypeptides, an intermediate class B is also designated (green). (C and D) Average H:L ratios (± SD) for the early, intermediate and late classes of MRPs for the LSU (C) and SSU (D), as identified in Table S1. The dashed line shows the H:L ratios expected for the rate of mitoribosome synthesis required to sustain a constant cellular complement of mitoribosomes in growing cells (see Supplemental Experimental Procedures). Some MRPs were not included in this analysis because of insufficient numbers of peptides or, for bL33m, atypically rapid accumulation (discussed in the text). See also Figures S1 and S2.
Figure 3. Mitoribosome Assembly Intermediates Containing Early-Binding Proteins Complete Assembly during Chase Incubation
(A and B) Scatterplots of the H:L ratios of individual LSU (A) and SSU (B) polypeptides, after the 4-hr pulse (ordinate) or after an additional 10-hr chase incubation (abscissa). Early- and late-binding proteins identified in Figure 2 are in red and blue, respectively, with intermediate LSU proteins in gold-green. (C and D) The early, intermediate, and late classes of assembled MRPs in the LSU (C) and SSU (D) show statistically significant differences in P4C10/P4, the ratio of H:L values after and before the chase. Here and in Figure 4E, the horizontal lines indicate the mean value for all data points. See also Table S1.
Figure 4. MRPs Are Synthesized and Imported in Excess and Can Be Unstable if Not Assembled
(A) The H:L ratio immediately after 4 hr of pulse labeling is greater for MRPs than for a standard set of mitochondrial proteins, indicating a higher rate of synthesis and mitochondrial import, but the H:L ratio of MRPs declines during a 10-hr chase to a value not significantly different from the stable standard proteins. Mean values are shown ± SD. (B) The H:L values observed for MRPs in assembled mitoribosomes before and after the chase are shown for comparison. The large SE after the pulse reflects variable assembly kinetics of individual proteins and is significantly smaller after the chase. Note the change in scale of the ordinate compared to (A). (C and D) The stability of LSU (C) and SSU (D) MRPs in early (red), intermediate (green), and late (blue) assembly classes is plotted against the H:L ratio after the pulse. Standard proteins are shown with open circles in both plots. (E) The stabilities (P4C10/expected) of early, intermediate, and late assembly classes of MRPs are significantly lower than those of the standard proteins, but not significantly different from one another. See also Tables S1 and S2.
Figure 5. Assembly Scheme for the SSU
, The early- and late-binding MRPs are illustrated in (A) and (B), respectively, from two points of view, one rotated 180° with respect to the other. The 12S rRNA is shown with light blue spheres representing individual residues. Proteins are shown as cartoon structures within transparent surfaces. (A) Early MRPs are shown in magenta and, for the group containing mS27, in red. uS5m is shown in a salmon color to indicate that it bridges the upper and lower groups, which are outlined by dashed circles and illustrated in greater detail in Figure S5. (B) Late-binding proteins are shown in blue along with the early proteins as in (A). (C) The assembly scheme for the SSU showing protein-protein interactions between individual or grouped polypeptides, with their longer standard names truncated to numbers for simplicity. Heavy dashed lines indicate interaction surface areas greater than 1,000 Å
2 (see Figure S4), while lighter dashed lines indicate interactions between 1,000 Å 2 and 350 Å 2. mS22, mS31, mS35, mS39, and bS1m are shown in boldface brown type, since they are early-binding proteins lacking extensive RNA contacts. Since they depend on other proteins for assembly, they may be considered secondary binding proteins associated with the indicated early clusters. uS6m and mS38 are shown as independent proteins with variable association with the mS26 group but did not yield sufficient proteomic data for definitive kinetic assignment.
Figure 6. Model for Assembly of the LSU
16S rRNA and tRNA
V are shown with spheres representing individual residues in gray or tan, respectively. Proteins are shown as cartoon structures within transparent surfaces. Major clusters of coordinately assembled interacting proteins are surrounded by dashed ovals and illustrated in Figures S6 and S7. (A) Early MRPs are shown in red, but with mL45 shown in magenta to provide a marker (B) Intermediate proteins are shown in green, but uL18m and mL63 are colored pea green, since they exhibit earlier binding kinetics than other intermediate proteins. (C) Late-binding proteins are shown in blue.
Figure 7. Scheme for Assembly of the LSU
, Assembly scheme showing protein-protein interactions between individual or grouped polypeptides with line designations based on Figure S4 as in Figure 5C and with the longer standard names of individual proteins truncated to numbers for simplicity. uL12m and uL1m are included in the early and intermediate groups based on the kinetic proteomic results but are outlined in red, since they were not identified in the cryo-EM structure. bL31m is shown as an atypical member of an early group that appears to join the structure at a later stage (see text for details). mL39, mL45, and mL50 are shown in boldface brown type, since they are early-binding proteins but do not have extensive RNA contacts. Since they depend on other proteins for assembly, they may be considered secondary binding proteins. mL38 and mL63 are shown in boldface red type, since their assembly kinetics resemble those of early-binding proteins, but they have very close associations with intermediate binding proteins. uL29m and bL34m are shown within a green ellipse associated with a late group, since they showed somewhat earlier assembly kinetics than other group members.
All figures (7)
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