Defining the interactome of the human mitochondrial ribosome identifies SMIM4 and TMEM223 as respiratory chain assembly factors

Human mitochondria express a genome that encodes thirteen core subunits of the oxidative phosphorylation system (OXPHOS). These proteins insert into the inner membrane co-translationally. Therefore, mitochondrial ribosomes engage with the OXA1L-insertase and membrane-associated proteins, which support membrane insertion of translation products and early assembly steps into OXPHOS complexes. To identify ribosome-associated biogenesis factors for the OXPHOS system, we purified ribosomes and associated proteins from mitochondria. We identified TMEM223 as a ribosome-associated protein involved in complex IV biogenesis. TMEM223 stimulates the translation of COX1 mRNA and is a constituent of early COX1 assembly intermediates. Moreover, we show that SMIM4 together with C12ORF73 interacts with newly synthesized cytochrome b to support initial steps of complex III biogenesis in complex with UQCC1 and UQCC2. Our analyses define the interactome of the human mitochondrial ribosome and reveal novel assembly factors for complex III and IV biogenesis that link early assembly stages to the translation machinery.


Sample-size estimation
• You should state whether an appropriate sample size was computed when the study was being designed • You should state the statistical method of sample size computation and any required assumptions • If no explicit power analysis was used, you should describe how you decided what sample (replicate) size (number) to use Please outline where this information can be found within the submission (e.g., sections or figure legends), or explain why this information doesn't apply to your submission:

Replicates
• You should report how often each experiment was performed • You should include a definition of biological versus technical replication • The data obtained should be provided and sufficient information should be provided to indicate the number of independent biological and/or technical replicates • If you encountered any outliers, you should describe how these were handled • Criteria for exclusion/inclusion of data should be clearly stated • High-throughput sequence data should be uploaded before submission, with a private link for reviewers provided (these are available from both GEO and ArrayExpress) Please outline where this information can be found within the submission (e.g., sections or figure legends), or explain why this information doesn't apply to your submission: Within this study we did not use mouse models or patient material, but rather based our study on the standard tissue culture HEK293T cell line. The cell line was tested on a regular basis for mycoplasma. The culturing of the HEK293T cells is described in detail in the material and methods section. Hence, sample size estimation was not required. For each experiment we used appropriate amounts of cells or mitochondria to guaranty a reliable readout of the experiments.
eLife Sciences Publications, Ltd is a limited liability non-profit non-stock corporation incorporated in the State of Delaware, USA, with company number 5030732, and is registered in the UK with company number FC030576 and branch number BR015634 at the address 1st Floor, 24 Hills Road, Cambridge CB2 1JP | August 2014 2 Each experiment was performed at least in triplicate as biological replicates. We define a biological replicate as by starting with the culturing of HEK293 T cells from a stock culture for each experiment individually. The stock of the HEK293T cells has been in culture for a maximum of three months. Hence, the designed experiment, such as siRNA applications, were performed with individually cultured HEK293T cells for each experiment. The obtained data are represented in the manuscript and if appropriate an statistical analysis has been performed, as it is stated within the figure legends. SEM quantification were used within the manuscript. Statistical quantifications were performed of at least three individual replicates as described in the Method section. Following good scientific practice, we included only data and figures with appropriate quality. If experiments did not have reveal a sufficient quality the experiment was repeated and not counted as replicate.

Statistical reporting • Statistical analysis methods should be described and justified
• Raw data should be presented in figures whenever informative to do so (typically when N per group is less than 10) • For each experiment, you should identify the statistical tests used, exact values of N, definitions of center, methods of multiple test correction, and dispersion and precision measures (e.g., mean, median, SD, SEM, confidence intervals; and, for the major substantive results, a measure of effect size (e.g., Pearson's r, Cohen's d) • Report exact p-values wherever possible alongside the summary statistics and 95% confidence intervals. These should be reported for all key questions and not only when the p-value is less than 0.05.
Please outline where this information can be found within the submission (e.g., sections or figure legends), or explain why this information doesn't apply to your submission: (For large datasets, or papers with a very large number of statistical tests, you may upload a single table file with tests, Ns, etc., with reference to sections in the manuscript.)

Group allocation
• Indicate how samples were allocated into experimental groups (in the case of clinical studies, please specify allocation to treatment method); if randomization was used, please also state if restricted randomization was applied • Indicate if masking was used during group allocation, data collection and/or data analysis Please outline where this information can be found within the submission (e.g., sections or figure legends), or explain why this information doesn't apply to your submission: Additional data files ("source data") • We encourage you to upload relevant additional data files, such as numerical data that are represented as a graph in a figure, or as a summary table If a statistical analysis has been appropriate we calculated SEM values. This is stated within the figure legends. Statistical analysis is described in the Method section. The statistical analysis of the mass spectrometry datasets are described in the material and method section as followed: The MS raw data were analyzed with MaxQuant/Andromeda (version 1.4.1.2 for mL62 FLAG and 1.5.5.1 for SMIM4 FLAG data; (Cox and Mann 2008;Cox et al. 2011)) and searched against the UniProt human proteome set including isoforms (release versions 08/2014 for mL62 FLAG and 08/2018 for SMIM4 FLAG data) using default settings except that the minimum requirements for protein identification and relative quantification were set to one unique peptide and one SILAC peptide pair, respectively. Arg10 and Lys8 were set as heavy labels. Carbamidomethylation of cysteine residues was considered as fixed, and N-terminal acetylation and oxidation of methionine as variable modifications. The options 'match between runs' and 'requantify' were enabled. The experiments were performed in four biological replicates including label-switch. Lists of proteins identified in the analyses of mL62 FLAG and SMIM4 FLAG complexes are provided in Supplementary Table S1 and S2, respectively and uploaded in the submission process.
Within this study we did not divide into experimental groups, since we used standard HEK293T cell for our experiments.