Mitochondrial proteostasis in the context of cellular and organismal health and aging

As a central hub of cellular metabolism and signaling, the mitochondrion is a crucial organelle whose dysfunction can cause disease and whose activity is intimately connected to aging. We review how the mitochondrial network maintains proteomic integrity, how mitochondrial proteotoxic stress is communicated and resolved in the context of the entire cell, and how mitochondrial systems function in the context of organismal health and aging. A deeper understanding of how mitochondrial protein quality control mechanisms are coordinated across these distinct biological levels should help explain why these mechanisms fail with age and, ultimately, how routes to intervention might be attained

Npl3 physically interacts with Bre1.

<p>Co-immunoprecipitation analyses of Npl3 and members of the histone H2B ubiquitination machinery. Whole cell extracts from strains with the indicated proteins endogenously tagged with HA or GFP were immunoprecipitated with an α-Npl3 antibody <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003101#pgen.1003101-Siebel1" target="_blank">[63]</a> or non-specific antibody (α–n.s.). Western blot using α-HA or α-GFP from each co-IP experiment is shown. The sensitivity of the interaction to RNase (lane 4, +RNaseA) was determined by treating lysates with RNase A prior to immunoprecipitation. Lane 1 shows 1/60 total sample for each lysate. Bottom panel confirms presence of Npl3 in the immunoprecipitate.</p

A chromatin-centered survey of Npl3 genetic interactions.

<p>Summary of chromatin and transcription factors that exhibit genetic interactions with a deletion of <i>NPL3</i>. Colored ovals represent subunits identified in the SGA screens or by directed genetics; red indicates a suppressive (positive) interaction and green indicates a synthetic (negative) interaction. Outlined ovals refer to the complex that individual subunits belong to. K123Ub refers to the <i>htb1K123R</i> point mutant. K36me refers the <i>hht1K36A</i> point mutant. Grey or white indicates the genetic interaction was not tested. Physical interactions tested by co-IP are Npl3:Bre1 (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003101#pgen-1003101-g003" target="_blank">Figure 3</a>) and Npl3:U1 <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003101#pgen.1003101-Kress1" target="_blank">[7]</a>. Bold rectangle indicates factors shown in this paper and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003101#pgen.1003101-Kress1" target="_blank">[7]</a> to promote splicing; whether the presence of Npl3 can influence local H2B ubiquitination levels or dynamics remains unresolved. The PolII C-terminal domain is drawn in grey.</p

Genetic interactions between H2B ubiquitination machinery and canonical splicing factors.

<p>(A) Synthetic growth analyses between <i>bre1Δ</i> and genes encoding splicing factors. Double mutants were generated by tetrad dissection; log-phase cultures of the indicated strains were serially diluted and grown at the indicated temperatures. To the right of panels is the name of the spliceosomal complex to which the splicing factor mutants belong. NTC: Nineteen Complex. (B) Growth analyses of <i>ubp8Δ</i> and genes encoding splicing factors. Double mutants were generated and analyzed as in (A).</p

Extensive negative genetic interactions with <i>npl3</i> Δ connect <i>NPL3</i> to chromatin biology.

<p>(A) Work flow for analysis of the integrated synthetic dataset. Synthetic genetic array technology was used to screen ∼4800 non-essential genes whose deletion conferred lethality to <i>npl3</i>Δ at 30°C. These results were augmented by including genes exhibiting a genetic interaction score of ≤−2.5 with <i>npl3</i>Δ <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003101#pgen.1003101-Wilmes1" target="_blank">[26]</a> and genes identified as synthetic sick or lethal using tetrad dissection and serial dilution (directed genetics). (B) Statistically significant negative interactions between <i>NPL3</i> and known complexes. Statistical analysis identified the indicated complexes as having subunits significantly enriched (P<0.05) in the integrated synthetic dataset. Size of circle is based on number of subunits whose deletion exacerbates the growth defect of <i>npl3</i>Δ and thickness of line scales with the significance of the enrichment. Circles are color-coded based on the biological process to which the complex belongs. (C) Synthetic growth analyses with <i>npl3</i>Δ and genes implicated in chromatin biology. Each panel shows a double mutant strain, cognate single deletions strains and a corresponding wild-type that have been serially diluted onto rich medium and grown at the indicated temperatures. Double mutants were isolated after tetrad dissection. To the right of panels is the name of the complex to which the single chromatin mutants belong. (D) Tetrad dissection analyses with <i>npl3</i>Δ. Tetrad dissection plates from the indicated crosses are shown with the inviable spore circled. Replica plating to infer genotype later showed that inviable spores are the double mutants. (E) Synthetic growth analyses with <i>npl3</i>Δ and genes encoding the H2B ubiquitination machinery. Shown are serial dilutions of the indicated strains after incubation at the indicated temperatures. Genotypes not originally tested in the SGA are <i>htb1K123R</i> and <i>bre1H665A</i>. The <i>BRE1</i> and <i>bre1H665A</i> strains contain a <i>bre1</i> deletion covered by a plasmid encoding the indicated <i>bre1</i> allele. Arrowheads refer to comparisons made in the text.</p

Splicing is sensitive to Npl3 and the H2B ubiquitination cycle.

<p>(A) Schematic of probes contained on the splicing microarray. (B) Splicing profile of single or double mutant strains compared to wild-type. Cultures of the indicated strains and isogenic wild-type strains were grown to mid-log phase at 30°C and shifted to the indicated temperature; cDNA from single and double mutant strains were competitively hybridized on the microarray against that from an isogenic wild-type. The heat map shows the log₂-ratio for each gene feature of the indicated strain compared to wild-type. Gene order along the y-axis is the same for all arrays. Transcripts that encode the ribosomal protein genes (RPGs) are highlighted in purple to the right of the heat maps. Data for example genes are replicated below the genome-wide heat map to show splicing defects and exacerbation of defects at individual RPGs and non-RPGs. (C) Histogram of log₂-based Intron Accumulation Index scores. An Intron Accumulation Index value was calculated for each intron-containing gene by normalizing the intron change to exon change (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003101#s4" target="_blank">Materials and Methods</a>). Histogram shows the number of genes with an Intron Accumulation Index score greater than 0.3. Heat map within histogram bars shows distribution of the severity of splicing defect.</p