Proteomic analysis of the ATP synthase interactome in notothenioids highlights a pathway that inhibits ceruloplasmin production

Abstract

Antarctic notothenioids have unique adaptations that allow them to thrive in subzero Antarctic waters. Within the suborder Notothenioidei, species of the family Channichthyidae (icefish) lack hemoglobin and in some instances myoglobin too. In studies of mitochondrial function of notothenioids, few have focused specifically on ATP synthase. In this study, we find that the icefish Champsocephalus gunnari has a significantly higher level of ATP synthase subunit α expression than the red-blooded Notothenia rossii, but a much smaller interactome than the other species. We characterize the interactome of ATP synthase subunit α in two red-blooded species Trematomus bernacchii, N. rossii, and in the icefish Chionodraco rastrospinosus and C. gunnari and find that, in comparison with the other species, reactome enrichment for C. gunnari lacks chaperonin-mediated protein folding, and fewer oxidative-stress-associated proteins are present in the identified interactome of C. gunnari. Reactome enrichment analysis also identifies a transcript-specific translational silencing pathway for the iron oxidase protein ceruloplasmin, which has previously been reported in studies of icefish as distinct from other red-blooded fish and vertebrates in its activity and RNA transcript expression. Ceruloplasmin protein expression is detected by Western blot in the liver of T. bernacchii, but not in N. rossii, C. rastrospinosus, and C. gunnari. We suggest that the translation of ceruloplasmin transcripts is silenced by the identified pathway in icefish notothenioids, which is indicative of altered iron metabolism and Fe(II) detoxification.

Keywords: ATP synthase; ceruloplasmin; mitochondria; notothenioid; proteomics.

Figure 1.

Western blot to measure the expression of ATP5A and ceruloplasmin. ATP5A in C. gunnari has significantly higher relative expression (ATP5A/GAPDH) than in N. rossii. Traces of ceruloplasmin expression were detected in T. bernacchii, but not in N. rossii, C. rastrospinosus, or C. gunnari. GAPDH present as a loading control. Error bars = means ± SE, Student’s unpaired t test *P < 0.05, n = 3. Samples are biological replicates. C. gunnari, Champsocephalus gunnari; C. rastrospinosus, Chionodraco rastrospinosus; N. rossii, Notothenia rossii; T. bernacchii, Trematomus bernacchii.

Figure 2.

Heatmaps of proteins detected by label-free mass spectrometry from coimmunoprecipitation samples. Samples are presented as mean relative percentage of total ion areas, an intensity-based quantification of relative protein abundance, from n = 3 biological replicates. A: relative percentage of total ion areas for all proteins identified across the four notothenioid species; B: proteins identified across all four species of notothenioid with q < 0.01 following multiway ANOVA analysis. C. gunnari, Champsocephalus gunnari; C. rastrospinosus, Chionodraco rastrospinosus; N. rossii, Notothenia rossii; T. bernacchii, Trematomus bernacchii.

Figure 3.

KEGG enrichment of the coimmunoprecipitated proteins for each of the four fish species: T. bernacchii (A), C. gunnari (B), C. rastrospinosus (C), and N. rossii (D). The five KEGG enriched terms with the lowest FDRs are the same for the four species: glycolysis/gluconeogenesis, biosynthesis of amino acids, carbon metabolism, ribosome, and metabolic pathways. The five pathways with the lowest FDRs within each species are scored according to strength of the enrichment effect [Log10(observed/expected)]. C. gunnari, Champsocephalus gunnari; C. rastrospinosus, Chionodraco rastrospinosus; FDR, false discovery rate; KEGG, Kyoto Encyclopedia of Genes and Genomes; N. rossii, Notothenia rossii; T. bernacchii, Trematomus bernacchii.

Figure 4.

Reactome enrichment of the proteins that co-immunoprecipitated with anti-ATP5A for each of the four notothenioid species: T. bernacchii (A), C. gunnari (B), C. rastrospinosus (C), N. rossii (D). The reactome enrichment terms with the lowest FDRs are varied between the four species, with only metabolism and L13a-mediated translational silencing of ceruloplasmin expression common to all four species. The five pathways with the lowest FDRs within each species are scored according to strength of the enrichment effect [Log10(observed/expected)]. C. gunnari, Champsocephalus gunnari; C. rastrospinosus, Chionodraco rastrospinosus; FDR, false discovery rate; N. rossii, Notothenia rossii; T. bernacchii, Trematomus bernacchii.

Figure 5.

PPI network of coimmunoprecipitated proteins in Trematomus bernacchii. Color of edges shows type of interactions. Color of nodes represents local network clusters. Cytoplasmic ribosomal proteins are red; carbon metabolism, and phosphoglycerate/bisphosphoglycerate mutase, and active site proteins are blue; mixed, incl. biosynthesis of amino acids, and one carbon pool by folate proteins are green; Pentose phosphate pathway, and glycolysis proteins are yellow; and mixed, incl. fatty acid degradation, and valine, leucine and isoleucine degradation proteins are pink. PPI, protein-protein interaction.

Figure 6.

PPI network of coimmunoprecipitated proteins in Champsocephalus gunnari. Color of edges shows type of interactions. Color of nodes represents local network clusters. Glycolysis and enolase conserved site proteins are red; cytoplasmic ribosomal proteins and elongation factor proteins are blue; and carbon metabolism and phosphoglycerate/bisphosphoglycerate mutase, active site proteins are green. PPI, protein-protein interaction.

Figure 7.

PPI network of coimmunoprecipitated proteins in Chionodraco rastrospinosus. Color of edges shows type of interactions. Color of nodes represents local network clusters. Cytoplasmic ribosomal proteins are red; carbon metabolism and phosphoglycerate/bisphosphoglycerate mutase, active site proteins are blue; mixed, incl. biosynthesis of amino acids, and one carbon pool by folate proteins are green; pentose phosphate pathway and glycolysis proteins are yellow; and mixed, incl. fatty acid degradation, and valine, leucine, and isoleucine degradation proteins are pink. PPI, protein-protein interaction.

Figure 8.

PPI network of co-immunoprecipitated proteins in Notothenia rossii. Color of edges shows type of interactions. Color of nodes represents local network clusters. Cytoplasmic ribosomal proteins are red; pentose phosphate pathway and glycolysis proteins are green; mixed, incl. biosynthesis of amino acids, and one carbon pool by folate proteins are blue; and mixed, incl. one-carbon metabolism, and glycine, serine and threonine metabolism proteins are yellow. PPI, protein-protein interaction.