Supplementary Data 1： ：The function analysis of every protein group or the very important mitochondrial proteins in INS-1 β cell
Metabolism In pancreatic β cells, insulin secretion is induced or enhanced by many metabolism-derived intermediates; therefore, metabolism-associated proteins take the central stage of the integrated mitochondrial function. Proteins assigned to this family are the most popular (49%) in all identified mitochondrial proteins, and it covers the most of basic substance metabolism in organisms as mentioned below. Most of proteins in the metabolism group are related with protein metabolism and modification. Mitochondria own a set of circle DNA that encodes some essential proteins of the respiratory chain, so there are a lot of proteins forming a complex translation system to regulate protein translation in mitochondria. 194 proteins, responsible for protein biosynthesis, post-translation modification (PTM) and proteolysis in mitochondria, were identified in our study. Among the 194 proteins, 19 proteins was annotated for amino acid activation, 93 proteins for protein biosynthesis, 19 proteins for protein folding, 16 proteins for PTM and 25 proteins for proteolysis. In this big group, some very important translational initiation factors and elongation factors for protein translation including mitochondrial translational initiation factor 2, eukaryotic translation elongation factor 2, G elongation factor, G elongation factor mitochondrial 2 and mitochondrial Tu translation elongation factor were presented here. These results indicated, at least, that this class of proteins in mitochondria would promote the synthesis of neonatal mitochondrial proteins continuously. The fast
protein synthesis of neonatal proteins could enhance the change of mitochondrial conformation to easily meet the mitochondrial functional requirements. Therefore, it proposed that both conformation and function of mitochondria were very active in β cell. Here, it is worthy to notice that we identified 76 ribosome proteins which belong to 28S, 30S, 39S, 40S, 50S and 60S subunits. The subunits of mitochondrial ribosomes are difficult to identify due to their low abundance in comparison with the overall mitochondrial proteome 1. So, the limited information is known about their roles in mitochondria
1, 2
. This work increases the number of mitochondrial ribosome
subunit identified. It would set a foundation for further investigation on the structure and function of mitochondrial ribosomes.
Lipid, fatty acid and steroid metabolism is another major category. 66 proteins were classified into this category, of which, 13 important enzymes for fatty acid beta-oxidation, 19 enzymes for fatty acid metabolism, 7 proteins for phospholipid metabolism, 3 proteins for lipid and fatty acid transport, 10 proteins for vitamin metabolism and 5 for steroid metabolism were found. In this group, many known membrane proteins were identified with high sequence coverage. For example, three key enzymes related to fatty acid oxidation, carnitine acetyltransferase, carnitine palmitoyltransferase 2 and liver carnitine palmitoyltransferase 1a (LCPTI), were identified with a sequence coverage of >40%. If this class of proteins were mapped into KEGG pathway, 25 proteins could be found in the pathway of fatty acid metabolism and these proteins cover the most of components of fatty acid metabolism
pathway (figure 3). Fatty acid metabolism is critical for insulin secretion 3. The alteration of these key enzymes of fatty acid metabolism or the disturbance of fatty acid metabolism can impair insulin secretion. For example, Herrero found that the overexpression of LCPTI could alter insulin secretion through interacting with increased malonyl-CoA 4. Mitochondria have their own mtDNA encoding some specific proteins. In this work, 65 proteins were classified into nucleoside, nucleotide and nucleic acid metabolism. These proteins in this category mainly participate in DNA replication, mRNA transcription, mRNA splicing, mRNA polyadenylation, tRNA or rRNA metabolism, nucleoside and nucleotide metabolism. These components set the prerequisite for mtDNA encoded mitochondrial proteins. Fourthly, 56 proteins participating in carbohydrate metabolism is another major category that is composed by various enzymes mainly including dehydrogenase, carbohydrate kinase, hydratase, acetyltransferase, glucosidase, epimerase/racemase, ligase and mutase. These enzymes are the crucial components of gluconeogenesis, glycolysis and tricarboxylic acid pathway (TCA). By KEGG, 18 proteins were match to TCA cycle, of which many proteins are the rate-limiting enzymes in TCA cycle (Supplementary figure 4). We have known that the metabolism of glucose in β cell provides many intermediates for the regulation of insulin secretion through TCA cycles 5. The detection of this protein class is helpful for further deciphering the roles of carbohydrate metabolic pathway in β cell insulin secretion. Finally, 41 proteins related to amino acid metabolism, 32 proteins related to coenzyme
and prosthetic group and some proteins involved in phosphate, nitrogen and sulfur metabolism etc, were found in this study. Many studies have proposed that the metabolism of some specific amino acids, including glutamate, glutamine and alanine, may also generate mitochondrial-derived messengers for insulin secretion
5-8
. The
turnover of glutamate in mitochondrial is regulated by glutamate dehydrogenase (GLUD). It has been reported that down-regulating GLUD activity could inhibit insulin exocytosis process by a mitochondrial ADP-ribosyltransferase SIRT4 9.
Electron transport Mitochondria produce ATP by electrons passing through mitochondrial respiratory complexes in the mitochondrial inner membrane. Up to date, the complete subunits of the electron transport chain remain unknown. In this study, 89 proteins involved in electron transport were identified. These proteins included oxidase, reductase, dehydrogenase and oxidoreductase, and mainly participated in acyl-CoA metabolism or/and oxidative phosphorylation. For example, acyl-Coenzyme A dehydrogenase complex were identified here with 7 subunits containing long-chain, medium chain, short chain, short/branched chain, very long chain, member 8, member 9 and member 10. In this work, many key components of mitochondrial respiratory complex I-V were included in this group. According to KEGG pathway analysis, 64 proteins were mapped to oxidative phosphorylation, of which we identified 29 separate polypeptide chains
of
NADH
dehydrogenase
(complex
I),
4
subunits
of
succinate
dehydrogenase(complex II), 6 protein subunits of cytochrome c reductase complex
(complex III), 8 subunits of cytochrome c oxidase (Complex IV) and 13 subunits of F-type ATPase (Complex V) (Supplementary figure 5 ). As been shown in STRING figure, these proteins tightly interact with other functional associated proteins.
Immunity and defense In this study, thirty proteins were classified into immunity and defense function family. Among the thirty proteins, nine proteins were annotated with the function of antioxidation and free radical removal, for example, hydroxyacyl glutathione hydrolase, peroxiredoxin 3, peroxiredoxin 5 and superoxide dismutase 2. We have known that mitochondria are not only the basic origin of ATP but also the major sites of reactive oxygen species (ROS). In general, ROS were produced through the electron leak on mitochondrial respiratory chain, and cleaned by the antioxidation system
10, 11
. The low concentration of ROS has their right physiological function on
mitochondria or cell. If this homeostasis is broken, mitochondrial functions are particularly susceptible to ROS due to the weak expression of natural enzymatic defence, e.g. SOD and catalase in β cell mitochondria
7, 8
. Redox stress has been
proven as the key culprit for the damage to mitochondria and the dysfunction of β cell 12, 13
. Because mitochondria are easy to exposure to redox stress, stress response
proteins sound very important for the self-protection of mitochondria
7, 8
. Here, nine
stress-response participating proteins were presented, for example, heat shock 70kD protein 5, heat shock 90kDa protein 1 beta, DJ-1 protein, TNF receptor-associated protein 1 and methionine sulfoxide reductase B2. Most of them also serve as
chaperones for protein folding. In addition, some proteins involved in immune response (ECSIT homolog, q subcomponent binding protein of complement component 1) and detoxification were presented by this study. Apparently, these protein expression patterns of defense group are beneficial for our understanding the self-protection mechanism of mitochondria.
Transport and Homeostasis As mitochondria are double membrane bounded compartments, substances shuttle frequently between mitochondria and cytosol or mitochondrial intermembrane space and matrix through the mitochondrial membrane transporters or translocases. By controlling the molecular flows, these transporters not only provide the precursors for metabolism, but also transmit the signals to maintain mitochondria or cellular dynamic homeostasis. 83 proteins were assigned to transport function group and five proteins are involved in cell homeostasis control. This number of proteins shown that, this class of proteins takes a big proportion of mitochondrial proteins. Most proteins of this class identified with high sequence coverage are ion channel or transfer/carrier proteins in charge of the transportation of ion hydrogen, calcium, potassium, sodium, magnesium, amino acid, phosphate, sulfur, nucleic acid or other small molecules. Eighteen proteins belongs to solute carrier family were detected, among them, fifteen proteins were classified into solute carrier family 7, including mitochondrial carnitine/acylcarnitine translocase, adenine nucleotide translocator, dicarboxylate transporter, glutamate carrier, phosphate carrier, oxoglutarate carrier etc. Notably,
three VADCs (voltage-dependent anion channel), VADC1, VADC2 and VADC3, were observed with coverage of >70%. VDAC, an outer mitochondrial membrane channel, allows diffusion of small hydrophilic molecules to keep the anion physiological homeostasis and energy metabolism, and may be responsible for the release of mitochondrial products that triggers apoptosis under the impairment of mitochondria 14-16
. In our results, three VDACs were identified in pancreatic β cell mitochondria,
which implicates every isoform own disticnt function in pancreatic β cell mitochondria. The apoptosis is the crucial cause for the reduction of β cell mass followed by insulin secretion decrease in diabetics. So, it is noteworthy to discover the different functions of VADC isoforms in pancreatic β cell mitochondria. In addition, uncoupling protein 2 (UCP-2), a very important mitochondrial protein in pancreatic β cell in diabetes genesis, also were detected although with a 6% sequence coverage. Mitochondria calcium as signal messenger raised many concerns gradually because of its many influences on mitochondria proteins and metabolism and further influences on β cell insulin secretion
17
. But it remains obscure about how calcium uptake into
mitochondria by the unknown calcium uniporters. Some studies also proposed VADC and UCP2 as calcium uniporters, but it is still controversial
17, 18
. In our study, Four
proteins, Atp1a1 (ATPase, Na+/K+ transporting, alpha 1 polypeptide), Atp1a2 (ATPase, Na+/K+ transporting, alpha 2 polypeptide), Atp2a2(ATPase, Ca++ transporting, cardiac muscle, slow twitch 2) and Atp2a3 (ATPase, Ca++ transporting, ubiquitous), identified with at least 15% sequence coverage, can regulate ion calcium
shuttle for keeping intracellular calcium homeostasis. If these proteins are authentic components of mitochondria, they may contribute to the uptake and balance of mitochondria calcium.
Intracellular protein traffic, Protein targeting and localization Transport of proteins within the extensive network of membrane-bound compartments is crucial for normal cell function in all eukaryotic cells. This process is highly regulated to ensure the specificity and efficiency of cargo delivery. Neonatal mitochondrial proteins encoded by nuclear genome need be transported into the specific location of mitochondria for their high performance on specific roles. Evidence has revealed that mitochondrial protein entry was directed to different import routes by various translocases, Such as TOMs in the mitochondrial outer membrane and TIMs in the mitochondrial inner membrane. We identified 49 proteins involved in intracellular protein traffic, including Tom20, Tom 22, Tom 40, Timm8a, Timm9, Timm9b, Timm10, Timm13, Timm17a, Timm17b, Timm22 and Timm44. Most of these translocase proteins were identified with high sequence coverage. The identification of new components of mitochondrial protein import machineries presented more clues for exploring the molecular mechanism of mitochondrial protein localization. Of course, it also raised many questions, e.g. how these components of entry machineries work together.
Signal transduction
As one key component of intact cell, mitochondria must communicate with other cellular components through different cellular signal transduction. Although these pathways have not been well elucidated yet to date, it has been recognized that mitochondrion is a critical signaling receiver/integrator
19
. Here, we identified some
signaling transduction proteins that were mainly classified into ras-, guanine- and GTP- related proteins, receptors and kinases /kinase inhibitors. Nine proteins belong to small GTPase, such as member RAS oncogene family RAB7, ras homolog gene family member T2, RAS related protein 1b, member RAS oncogene family RAB14. GTPase proteins may form a link between protein kinases and mitochondrial protein effectors. Recently, many studies have proposed that GTPase family proteins were involved in the governance of mitochondrial dynamics including fission, fusion and structural change
20
. The aberration of mitochondrial dynamics is accompanied with
the damage of mitochondrial metabolism
20
. These structural changes in β cell
mitochondria would influence insulin secretion of β cells.
Apoptosis Pancreatic β cell apoptosis is a pivotal cause for the decrease of β cell mass and gross insulin secretion
21
. In our study, sixteen important proteins involved in apoptosis
were identified. Most of them are the key components of apoptosis pathway, including programmed cell death 8 (PCD8, also known as apoptosis-inducing factor), BCL2-like 1,BCL2-like 11, BCL2-antagonist/killer 1(Bak1), Bcl2-associated X protein(Bax), endonuclease G, endonuclease G-like 1, cytochrome c, estrogen
receptor-binding fragment-associated gene 9, FK506 binding protein 8 and myeloid cell leukemia sequence 1.
Cell cycle, Cell proliferation, differentiation and other functions Nine mitochondrial proteins assigned to this class are related to DNA replication. Such as mitochondrial topoisomerase I, polymerase (DNA directed) gamma, progressive external ophthalmoplegia 1 homolog, prohibitin 2, prohibitin. By controlling DNA replication or protein biosynthesis, they may influence cell cycle, cell proliferation and differentiation. Twenty-one proteins were classified into the functional group of developmental processes. Protein mitofusin 1 and mitofusin 2, two essential transmembrane GTPases that mediate mitochondria fusion and fission, play important roles in the mitochondrial conformation
22
. Intermediate filament
peripherin 1 and receptor protein basigin have specific roles in ectoderm developmental process. Besides that, seventeen cell structure related proteins were annotated or predicted in mitochondria in our study. Among these proteins, nine proteins are intermediate filament. In addition, the biological processes of 175 proteins were unclassified.
References 1.
Koc, E. C.; Burkhart, W.; Blackburn, K.; Moseley, A.; Koc, H.; Spremulli, L. L., A proteomics
approach to the identification of mammalian mitochondrial small subunit ribosomal proteins. J Biol Chem 2000, 275, (42), 32585-91.
2.
Bolender, N.; Sickmann, A.; Wagner, R.; Meisinger, C.; Pfanner, N., Multiple pathways for
sorting mitochondrial precursor proteins. EMBO Rep 2008, 9, (1), 42-9. 3.
Yaney, G. C.; Corkey, B. E., Fatty acid metabolism and insulin secretion in pancreatic beta cells.
Diabetologia 2003, 46, (10), 1297-312. 4.
Herrero, L.; Rubi, B.; Sebastian, D.; Serra, D.; Asins, G.; Maechler, P.; Prentki, M.; Hegardt, F. G.,
Alteration of the malonyl-CoA/carnitine palmitoyltransferase I interaction in the beta-cell impairs glucose-induced insulin secretion. Diabetes 2005, 54, (2), 462-71. 5.
Newsholme, P.; Bender, K.; Kiely, A.; Brennan, L., Amino acid metabolism, insulin secretion and
diabetes. Biochem Soc Trans 2007, 35, (Pt 5), 1180-6. 6.
Eliasson, L.; Abdulkader, F.; Braun, M.; Galvanovskis, J.; Hoppa, M. B.; Rorsman, P., Novel
aspects of the molecular mechanisms controlling insulin secretion. J Physiol 2008, 586, (14), 3313-24. 7.
Maechler, P.; Carobbio, S.; Rubi, B., In beta-cells, mitochondria integrate and generate metabolic
signals controlling insulin secretion. Int J Biochem Cell Biol 2006, 38, (5-6), 696-709. 8.
Maechler, P.; de Andrade, P. B., Mitochondrial damages and the regulation of insulin secretion.
Biochem Soc Trans 2006, 34, (Pt 5), 824-7. 9.
Ahuja, N.; Schwer, B.; Carobbio, S.; Waltregny, D.; North, B. J.; Castronovo, V.; Maechler, P.;
Verdin, E., Regulation of insulin secretion by SIRT4, a mitochondrial ADP-ribosyltransferase. J Biol Chem 2007, 282, (46), 33583-92. 10. Brookes, P. S.; Levonen, A. L.; Shiva, S.; Sarti, P.; Darley-Usmar, V. M., Mitochondria: regulators of signal transduction by reactive oxygen and nitrogen species. Free Radic Biol Med 2002, 33, (6), 755-64. 11. Brand, M. D.; Affourtit, C.; Esteves, T. C.; Green, K.; Lambert, A. J.; Miwa, S.; Pakay, J. L.;
Parker, N., Mitochondrial superoxide: production, biological effects, and activation of uncoupling proteins. Free Radic Biol Med 2004, 37, (6), 755-67. 12. Maechler, P.; Wollheim, C. B., Mitochondrial function in normal and diabetic beta-cells. Nature 2001, 414, (6865), 807-12. 13. Mulder, H.; Ling, C., Mitochondrial dysfunction in pancreatic beta-cells in Type 2 diabetes. Mol Cell Endocrinol 2009, 297, (1-2), 34-40. 14. Rasola, A.; Bernardi, P., The mitochondrial permeability transition pore and its involvement in cell death and in disease pathogenesis. Apoptosis 2007, 12, (5), 815-33. 15. Shoshan-Barmatz, V.; Keinan, N.; Zaid, H., Uncovering the role of VDAC in the regulation of cell life and death. J Bioenerg Biomembr 2008, 40, (3), 183-91. 16. Colombini, M., VDAC: the channel at the interface between mitochondria and the cytosol. Mol Cell Biochem 2004, 256-257, (1-2), 107-15. 17. Wiederkehr, A.; Wollheim, C. B., Impact of mitochondrial calcium on the coupling of metabolism to insulin secretion in the pancreatic beta-cell. Cell Calcium 2008, 44, (1), 64-76. 18. Gincel, D.; Zaid, H.; Shoshan-Barmatz, V., Calcium binding and translocation by the voltage-dependent anion channel: a possible regulatory mechanism in mitochondrial function. Biochem J 2001, 358, (Pt 1), 147-55. 19. Goldenthal, M. J.; Marin-Garcia, J., Mitochondrial signaling pathways: a receiver/integrator organelle. Mol Cell Biochem 2004, 262, (1-2), 1-16. 20. McBride, H. M.; Neuspiel, M.; Wasiak, S., Mitochondria: more than just a powerhouse. Curr Biol 2006, 16, (14), R551-60. 21. Chandra, J.; Zhivotovsky, B.; Zaitsev, S.; Juntti-Berggren, L.; Berggren, P. O.; Orrenius, S., Role
of apoptosis in pancreatic beta-cell death in diabetes. Diabetes 2001, 50 Suppl 1, S44-7. 22. Ishihara, N.; Eura, Y.; Mihara, K., Mitofusin 1 and 2 play distinct roles in mitochondrial fusion reactions via GTPase activity. J Cell Sci 2004, 117, (Pt 26), 6535-46.
