Proteomics of Bovine Mitochondrial RNA-Binding Proteins: HES1/KNP-I Is a New Mitochondrial Resident Protein Mikhail V. Ponamarev,† Yi-Min She,‡ Li Zhang,‡ and Brian H. Robinson*,† Department of Metabolism and Department of Structural Biology and Biochemistry, Hospital for Sick Children Research Institute, Toronto M5G 1X8, Ontario, Canada Received July 26, 2004
Proteomic analysis of bovine mitochondrial proteins with affinity to polyAdenylate or polyUridylate was performed in an effort to identify novel RNA-binding mitochondrial proteins. We have used 2D gel electrophoresis and MALDI-QqTOF mass spectrometry to identify a total of 64 proteins, of which 51 have defined mitochondrial function including 6 known RNA-binding proteins. HES1/KNP-I from the polyA-binding fraction of mitochondrial Triton extract showed exclusive mitochondrial localization when expressed in GFP-tagged form. The HES1/KNP-I gene is on human chromosome 21q22.3 and may be involved in several disorders mapped to that region. Thus, HES1/KNP-I is a proven mitochondrial resident protein with apparent tight membrane association and tentative RNA-binding properties. Keywords: RNA • mitochondria • 2D • HES1 • proteomics • ζ-crystallin
Introduction To gain a complete understanding of the critical role of mitochondria in cell function and human diseases, we must compile a list of all proteins involved in mitochondrial function. This is a challenging task due to the diversity of the mitochondrial proteome across tissues and to difficulties in identifying mitochondrial low abundance proteins. Several groups have undertaken thorough studies to characterize the mitochondrial proteome in different species, including human and mouse. A recent comprehensive yeast mitochondrial proteomics study reported 750 identified proteins which is thought to represent about 90% of the yeast mitochondrial proteome.1 It provides a good foundation for future characterization of novel mitochondrial functions since the biochemical role for a quarter of identified proteins remains unidentified. To better understand the molecular basis for many human diseases associated with mitochondrial dysfunction, the comprehensive description of the mitochondrial proteome in humans and model mammalian organisms is critical. In the human heart mitochondrial proteomic study, 615 distinct proteins were identified with functions in oxidative phosphorylation; signaling; RNA, DNA, and protein synthesis; ion transport; apoptosis; lipid metabolism; protein processing; and targeting.2 117 proteins had no assigned function. These data were combined with the data from public sequence databases to create the MitoProteome database that lists 847 human mitochondrial proteins.3 A mouse mitochondrial proteomic survey resulted in a list of 591 proteins, with 163 proteins not * To whom correspondence should be addressed: Dr. Brian Robinson, Program Head, Metabolism, Research Institute, Hospital for Sick Children, 555 University Ave, Toronto, ONTARIO, M5G 1X8 Canada. Tel: (416) 8135989. Fax: (416) 813-8700. E-mail:
[email protected]. † Department of Metabolism. ‡ Department of Structural Biology and Biochemistry. 10.1021/pr049872g CCC: $30.25
2005 American Chemical Society
previously defined as mitochondrial.4 The study also included RNA expression analysis, Green fluorescent protein (GFP)tagging experiments, data on tissue-specific differences in the mitochondrial proteome and bioinformatic analysis of correlations in protein expression patterns to provide an independent evidence for protein mitochondrial localization and a better insight into mitochondrial biogenesis, range of functions and evolution. MITOPRED (http://mitopred.sdsc.edu), the new robust method in predicting the nucleus-encoded mitochondrial proteins, was used to analyze six complete genomes (3 invertebrate, 2 vertebrate and 1 plant species) and to estimate the total number in each genome. In humans, this method predicts the existence of 1362 mitochondrial proteins corresponding to 4.8% of the total proteome.5 The goal of the present mitochondrial proteomics project was to identify novel proteins or protein isoforms localized to the mitochondria. We utilized ribohomopolymer affinity chromatography to reduce protein sample complexity and enrich it for the proteins with a specific property of affinity to RNA. The requirement for large amounts of mitochondria needed to enrich the sample for low abundance proteins dictated the choice of beef heart as a starting material. Two different methods were used to extract soluble proteins from mitochondria. PolyAdenylate (polyA) and polyUridylate (polyU) affinity matrixes, used in this study, were expected to bind (1) proteins with specific affinity for polyA sequence, (2) polyA-tailed mitochondrial transcription products with associated proteins, as well as (3) proteins with general RNA- or DNA-binding properties. Mitochondrial RNA-binding proteins identified in previous proteomic studies included mitochondrial ribosomal proteins, tRNA synthetases, AuH protein (RNA binding homologue of Coenzyme A hydratase), splicing factor 2 (SF2)- associated p32 protein, leucine rich PPR (pentatricopeptide) motif containing Journal of Proteome Research 2005, 4, 43-52
43
Published on Web 01/21/2005
research articles protein (LRPPRC) and ribonuclease H1.2,4 UV-cross-linking experiments on mitochondrial RNA-binding proteins from beef liver identified a 78 kDa protein, associated with the mitochondrial ribosome 28S small subunit, as a homologue of human 689 aa PPR motif containing protein and three others were hypothesized to be glutamate dehydrogenase, AuH protein (RNA-binding homologue of Coenzyme A hydratase) and a putative mammalian homologue of yeast mitochondrial helicase SUV3.6 The precedent for studying mitochondrial RNAbinding proteins and their critical role in mitochondrial function is predicated on the recent discovery of the mutations in LRPPRC protein responsible for human cytochrome c oxidase deficiency in the French Canadian variant of Leigh syndrome.7
Experimental Section Procedures. 1. Mitochondria Preparation. Fresh beef heart mitochondria were isolated as previously described.8 Briefly, the tissue was ground, by subjecting chopped portions to a Waring blender, then homogenized in 50 mM HEPES buffer pH 7.4, 70 mM sucrose, 220 mM mannitol, 1 mM EGTA and 1 mg/mL fatty acid free BSA (Sigma-Aldrich, St. Louis, MO). The mixture was centrifuged at 3000 × g for 10 min to remove cell debris. The supernatant was centrifuged at 7000 × g for 20 min. The mitochondrial pellet was resuspended in homogenization buffer and again centrifuged at 7000 × g for 20 min. The pellet was resuspended in homogenization buffer to a final protein concentration of approximately 3 mg/mL, divided into 30 mL aliquots and snap-frozen in liquid nitrogen for storage at -70 °C. 2. PolyA/PolyU Sepharose Chromatography and Protein Sample Preparation. Two matrixes, polyA-Sepharose and polyU-Sepharose (Amersham Pharmacia), were used to enrich the sample for proteins with RNA-binding properties. Two different magnesium concentrations (2 mM and 10 mM) were used in separate chromatography experiments designed to modulate protein-RNA interaction. To obtain a sample of soluble mitochondrial proteins, mitochondria (200 mLs, ∼600 mg of protein) were extracted 5 times by homogenizing in buffer A (25 mM Tris-HCl pH 7.0, 50 mM NaCl, 1 mM EDTA, 2 mM MgCl2, 10% glycerol, 1 mM β-mercaptoethanol, 100 µg/mL BSA) and sonicating (sample volume 20-25 mLs, output power 10 W, 4 pulses for 5 s, 60 Sonic Dismembrator, Fisher Scientific). Supernatants (20 000 × g for 20 min) from all extractions were pooled together. These centrifugation steps have also ensured that contaminating membranes and associated proteins would stay in the pellet. 1 and 0.5 g of polyA-Sepharose and polyU-Sepharose respectively were equilibrated in buffer B (10 mM Tris-HCl pH 7.5, 50 mM NaCl, 1 mM EDTA, 2 mM MgCl2, 0.1% NP-40) and loaded on the Econo-Pac (Bio-Rad) 1.5 × 12 cm polypropylene columns at 4 °C. Mitochondrial extract was passed twice sequentially through polyU-Sepharose and polyA-Sepharose columns. Columns were washed with 50 volumes of buffer B. Bound proteins were eluted by applying sequentially 1.5 mL of 10 mM Tris-HCl, pH 7.5, 50% formamide and 2 mLs of 10 mM Tris-HCl, pH 7.5, 0.5 M NaCl to polyU-sepharose column or 3 mL of 10 mM Tris-HCl, pH 7.5, 0.5 M NaCl and 2 mLs of 10 mM Tris-HCl, pH 7.5, 50% formamide to polyA-sepharose column. Proteins were precipitated by adding 4 volumes of absolute ethanol and incubating samples overnight at -80 °C. The same experiment was repeated with buffers A and B containing 10 mM MgCl2. Mg2+ ions can stimulate or inhibit 44
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complex formation of proteins with nucleic acids depending on concentration. To deplete the sample of well studied high abundance proteins and to characterize soluble proteins with tighter membrane association that have affinity to polyA, the sample was prepared as follows. Mitochondria (120 mL, ∼360 mg of protein) were extracted 4 times by homogenizing in buffer A and sonicating. Soluble fractions were discarded. Final pellet was extracted by homogenizing in buffer A plus 1 M NaCl, stirring for 15 min at 4 °C and centrifuging at 20 000 × g. The pellet was extracted once more by homogenizing in buffer A plus 100 µg/mL BSA, 5 mM MgCl2, 1.6% Triton and stirred for 15 min at 4 °C, and centrifuged at 20 000 × g. Soluble fractions from two last extractions were dialyzed against 25 mM TrisHCl pH 7.2, 100 mM NaCl, 5% glycerol, 1 mM β-mercaptoethanol, 5 mM MgCl2 (plus 0.1% Triton in the case of the Triton extraction fraction) overnight. PolyA column chromatography and protein sample preparation were performed as described above. 3. 2-D Gel Electrophoresis, Mass Spectrometry and Data Processing. Precipitated proteins were spun down, redissolved in rehydration buffer, and dialyzed against the latter for 1 day with two changes of buffer. Rehydration, sample dialysis and strip equilibration buffers were freshly made before each experiment using ultrapure grade urea (Invitrogen) to prevent formation of protein-urea adducts that can alter pI and molecular mass of polypeptides. Any heating of protein samples was avoided. Electrofocusing (Immobiline dryStrip pH range 3-10 NL, Ettan IPGphor, Amersham Biosciences) and second dimension gel electrophoresis (Protean II xi Cell, 16 × 20 cm, 1.5 mm thick gel, BioRad) were run according to manufacturers protocols. IPG strip rehydration buffer contained 8M urea, 2% CHAPS, 0.5% IPG buffer and 0.002% bromophenol blue. Prior to second dimension electrophoresis IPG strips were equilibrated for 15 min in 2% SDS, 50 mM Tris-HCl pH 8.8, 6 M Urea, 30% glycerol, 0.002% bromophenol blue, 10 mg/mL DTT followed by 15 min equilibration in the same buffer without DTT in the presence of 25 mg/mL of iodoacetamide. 2D-Gels were stained with ProteomIQ Blue Gel Stain (Proteome Systems Ltd.), a reformulated colloidal Coomassie blue stain that provides better sensitivity than traditional Coomassie staining. Protein spots excised from the gels were subjected to tryptic digestion and processed for MALDI on the ZipPlate (Millipore) according to the manufacturer protocol (available from Millipore website www.Millipore.com). In-gel trypsin digestion was achieved by adding 15 µL of trypsin buffer (11 µg/mL of porcine trypsin (Roche) in 25 mM ammonium bicarbonate) per 1 well of ZipPlate with excised gel piece and incubating overnight at 30 °C. In-gel digests were analyzed by MALDI MS (peptide mapping) and MS/MS (peptide sequencing) measurements on the Applied Biosystems/MDS Sciex QSTAR XL matrix-assisted laser desorption ionization quadrupole time-of-flight (MALDIQqTOF) mass spectrometer at The Hospital for Sick Children, Toronto. The instrument was equipped with a UV nitrogen laser (337 nm), and an acceleration voltage of 4 kV was used. 2,5dihydroxybenzoic acid (DHB) was used as the matrix. Nitrogen and argon were used as curtain and collision gases, respectively. Monoisotopic peptide masses were used to search the NCBI protein database with Protein Prospector algorithm (http:// prospector.ucsf.edu) MS-Fit. Protein identity was further validated by tandem MS/MS measurements on the selected peptide ions, and subsequently searched by MS-Tag. The mass tolerance for both parent and fragment ions was set to 50 ppm,
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Proteomics of Mitochondrial RNA-Binding Proteins
Table 1. List of the Bovine Mitochondrial Proteins Bound to PolyA- or PolyU- Sepharose and Identified by In-Gel Tryptic Digestion, MALDI MS and MS/MS, Database Search with Prospector MS-Fit and MS-Taga
annotation
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 51. 52. 53. 54. 55. 56.
actin R, cardiac myosin, β, heavy chain myosin light chain 3, cardiac tropomyosin 1, R vimentin troponin T glyceraldehyde 3-phosphate dehydrogenase fructose-1,6-bisphosphate aldolase A heat shock 27kDa protein heat shock 60kDa protein heat shock 70kDa protein isocitrate dehydrogenase, R isocitrate dehydrogenase,β isocitrate dehydrogenase, γ isocitrate dehydrogenase, (NADP+ dependent) malate dehydrogenase oxoglutarate dehydrogenase (lipoamide) dihydrolipoamide S-succinyltransferase, OGDH-E2 glutamate dehydrogenase aconitase 2, mitochondrial dihydrolipoamide dehydrogenase, PDH-E3 pyruvate dehydrogenase (PDH), E1-R pyruvate dehydrogenase (PDH), E1-β NADH dehydrogenase, CI-β 22kDa NADH dehydrogenase, CI-PDSW NADH dehydrogenase, CI-23kDa (TYKY) NADH dehydrogenase, CI-30kDa NADH dehydrogenase, CI-42kDa NADH dehydrogenase, CI-49kDa NADH dehydrogenase, CI-51kDa NADH dehydrogenase, CI-75kDa succinate dehydrogenase, subunit B, iron-sulfur succinate dehydrogenase, flavoprotein bc1 complex, core protein I bc1 complex, core protein II bc1 complex, Rieske ISP ATP synthase, R ATP synthase, β ATP synthase, δ ATP synthase, O subunit (OSCP) ETF-ubiquinone oxidoreductase acetyl-Coenzyme A C-acetyltransferase/thiolase dihydrolipoamide S-(2-methylpropanoyl) transferase branched chain keto acid DH E1-R branched chain keto acid dehydrogenase E1-β acyl-Coenzyme A DH, short-chain specific 3-hydroxyacyl-Coenzyme A DH, type II dehydrogenase/3-ketoacyl-Coenzyme A thiolase/ enoyl-coenzyme A hydratase, R dehydrogenase/3-ketoacyl-Coenzyme A thiolase/ enoyl-coenzyme A hydratase, β ζ-Crystallin/quinone reductase AuH, RNA binding homologue of CoA hydratase adenilate kinase 3 creatin kinase, muscle cardiac calsequestrin voltage dependent anion channel 1(VDAC1) apoptosis inducing factor
kDa/pI
matched peptides
sequence coverage (%)
71616 29468 4557777 136092 27806785 2119374 2506439 6730618 1170366 11560024 12653415 27807161 6166245 1170481 28461205 65932 4505493 21313536 118533 27806769 1706444 448580 18152793 28461191 28461255 27807359 128860 28603782 833783 548387 27807355 9257242 284648 27807137 27807143 1351360 27807237 28461221 27807305 27806307 4758312 86728 27806905 27806229 27806223 1168286 27805907 27805909
42/5.2 134/5.2 22/5.0 33/4.7 54/5.2 34/5.3 34/8.7 39/8.2 23/6.2 60/5.4 74/5.9 36/5.7 42/9.2 40/9.0 51/9.0 33/9.0 113/6.9 48/9.1 56/8.3 85/8.1 54/7.5 40/6.8 39/6.4 22/8.8 22/8.8 24/6.3 30/6.5 39/6.5 49/5.9 51/8.4 79/5.8 31/9.1 72/7.4 53/5.9 48/8.8 30/9.0 59/9.2 56/5.1 18/6.0 23/9.9 68/7.3 45/9.1 53/8.5 52/8.6 43/5.7 45/8.5 27/8.4 85/9.2
13 24 8 22 8 8 7 7 8 15 15 15 12 7 18 13 21 15 12 10 14 12 8 12 11 7 19 8 17 20 27 10 14 21 13 6 34 18 9 9 13 16 9 22 8 10 8 18
39 26 40 65 18 33 21 24 38 31 27 35 27 32 42 52 31 28 31 18 42 25 26 50 31 35 57 31 52 60 40 35 32 51 36 25 55 31 63 35 29 58 22 58 30 32 57 33
structural structural structural structural structural structural glycolysis glycolysis stabilization stabilization stabilization TCA cycle TCA cycle TCA cycle TCA cycle TCA cycle TCA cycle TCA cycle TCA cycle TCA cycle TCA cycle TCA cycle TCA cycle complex I complex I complex I complex I complex I complex I complex I complex I complex II complex II complex III complex III complex III complex V complex V complex V complex V redox lipid metabolism lipid metabolism lipid metabolism lipid metabolism lipid metabolism lipid metabolism lipid metabolism
BT
27885005
51/9.4
8
24
lipid metabolism
BT HS BT HS HS BT HS
27806685 4502327 27806319 21536288 4557409 27806185 095831
35/8.3 35/9.5 25/9.0 42/6.6 46/4.2 31/8.6 68/9.0
15 10 18 8 9 13 14
46 23 54 31 21 59 29
mRNA stability mRNA stability nucleotide metabolism nucleotide metabolism transport channels apoptosis
species
accession no.
BT HS HS HS BT BT BT OC CF RN HS BT BT MF BT SS HS MM BT BT CF SS MM BT BT BT BT BT BT BT BT HS BT BT BT BT BT BT BT BT HS MM BT BT BT RN BT BT
function
a Molecular weight and pI are shown for protein sequences in the NCBI database. Species: BT- Bos taurus, bovine; HS - Homo sapiens, human; RN Rattus norvegicus, rat; OC - Oryctolagus cuniculus, rabbit; SS - Sus scrofa, pig; CF - Canis familiariz, dog; MM - Mus musculus, mouse.
allowing possible post-translational modifications of carbamidomethylation on the cysteine residues. The number of unmatched ions was set to a maximum of 30%. If there was no reasonable hit in the NCBI protein database, then MS-Tag searching was then performed on the Bos taurus EST database. The obtained peptide sequences were finally searched against the human or mouse protein homology database with
Basic Local Alignment Search Tool (BLAST) (http:// www.ncbi.nlm.nih.gov:80/BLAST/). 4. Mitochondrial Localization by GFP Fusion and Microscopy. The target gene was amplified from normal fibroblasts cDNA by PCR using HiFi Taq polymerase (Invitrogen) and specific primers. PCR products of appropriate size were cloned into pCR2.1-TOPO vector (Invitrogen) and sequenced. The gene Journal of Proteome Research • Vol. 4, No. 1, 2005 45
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Table 2. List of the Bovine Mitochondrial Proteins Identified by In-Gel Tryptic Digestion, MALDI MS and MS/MS, EST Database Search with Prospector MS-Tag Followed by NCBI Protein Database Search by BLASTa annotation
57. myosin binding protein C
58. collagen R-1 (VI)
59. estradiol 17 β-dehydrogenase 8
60. 2,4-dienoyl-CoA reductase
61. HES1/KNP-I
62. pre-mRNA splicing factor SF2p32
63. carnitine acetyltransferase
46
m/z (meas.)
[MH]+ (calcd.)
1027.543 1256.671
1027.546 1256.675
1543.836
1543.833
1669.843 1850.971
1669.844 1850.965
1216.711
1216.705
1255.605
1255.596
1162.620
1162.604
1628.779
1628.778
1772.852
1772.860
1784.857
1784.879
2674.370
2674.381
1112.639 1357.709
1112.647 1357.712
1868.914
1868.903
2320.209
2320.205
1369.644
1369.642
1856.956
1856.966
2744.355
2744.364
1483.677
1483.671
1607.748
1607.737
1669.825
1669.833
1033.599 1619.811
1033.593 1619.803
1728.887
1728.881
1762.001
1761.990
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peptide sequence
accession no.
kDa
YIFESIGAK IAFQHGVTDLR ....+...... iafqygvtdlr TNGGVSVAELIVQEK .+.. ++........ tsggqalaelivQEK LEAPAEEDVWEILR WLKDGVELTREETFK
2920823
141
AGVEIFAVVVGR ..+... ..... AGIEIFVVVVG AAEYDVVFGER ..... .... TAEYDVAFGER CNSVLPGFIR ......... CNSVLPGFIA GAHAAFQADVSEAGAAR . .........+ ... GKHAAFQADVSQGPAAR LAAEGAAVAACDLDGAAAR .................. LAAEGAAVAACDLDGAAAQ GAHAAFQADVSEAGAARR . .........+ .... GKHAAFQADVSQGPAARR LLEQVQACFSRPPSVVVSCAGITR ...+.................... LLEEVQACFSRPPSVVVSCAGITR FNVIQPGPIK DQWDIIEGLIR +..... ... EQWDTIEELIR FDGGEEVLISGEFNSLR .... .......... FDGGGEVLISGEGNDLR VIGHPDIVINNAAGNFISPSER . ...+.............+.. vaghpnivinnaagnfispter NLSTFAVDGGTCK ........... NLSTFAVDGKDCK LTAVNHDAAIFPGGFGAAK .+. ............... LSAANHDAAIFPGGFGAAK GVEVTVGHEQEEGGKWPHAGTAEVIK .................+..... +. GVEVTVGHEQEEGGKWPYAGTAEAMK EVSFQATGESDWK .....+....+.. EVSFQSTGESEWK MSGGWELEVNGTEAK ........+...... MSGGWELELNGTEAK AFVDFLSDEIKEEK .............+ AFVDFLSDEIKEER LIEGVLDFK QLVEEFQTAGGVGER ...+... +...... QLVDEFQASGGVGER ALQPIVSEEEWAQTK .............. ALQPIVSEEEWAHTK LPVPPLQQTLDHYLK ........+...... LPVPPLQQSLDHYLK
6753484
108
7305125
28
1575000
36
1655594
28
338043
31
30582441
62
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Proteomics of Mitochondrial RNA-Binding Proteins Table 2. (Continued) annotation
64. acyl-Coenzyme A dehydrogenase
m/z (meas.)
[MH]+ (calcd.)
peptide sequence
accession no.
kDa
1079.555 1286.675
1079.548 1286.686
4501859
47
1350.708
1350.706
1478.799
1478.801
1763.905
1763.900
2077.107
2077.100
2386.170
2386.171
YAIGSLNEGR TNILGQVGHGYK .....+..... ANILGQIGHGYK FAQEQVAPFVSK .....+.... FAQEQIAPLVST KFAQEQVAPFVSK ......+.... KFAQEQIAPLVST ASSTCPVTFENVKVPK ......+........ ASSTCPLTFENVKVPE SVIQGLFQQGLMGIEIDTK ...............+. SVIQGLFQQGLMGIEVDPE GITCFLVDGDTEGLHVGKPENK ... ...... ...+...... GITSFLVDRDTPGLHIGKPENK
a The measured and calculated peptide masses are shown as well as single peptide sequence if identical between species or otherwise aligned with a homologous sequence from human or mouse protein in the NCBI database.
was amplified from the Topo vector using T7 promoter forward primer and a gene-specific reverse primer, which contained a BamHI site to ensure inframe fusion with N-terminal GFP. The product was digested with EcoRI/BamHI and cloned into pEGFP-N1 vector (BD Biosciences Clontech). Control fibroblast cells that have been immortalized using the large T antigen of SV40 or COS-1 cells were transfected with plasmid DNA using Qiagen Superfect reagent. After selection with Geneticin, cells were subcultured and subjected to fluorescence microscopy. Images were acquired either by confocal microscope Nikon Eclipse E1000 (60x objective) or by RTM-3 microscope (Richardson Technology Inc., Bolton, Canada) with a water immersion 63× objective.
Results and Discussion Beef Heart Mitochondria as the Source for Proteomic Study. Beef heart was the material of choice for this proteomic study because it provided the large amount of purified mitochondria needed to enrich the sample for low abundance proteins and to overcome the low sensitivity of the experimental setup. However, a significant limitation of using the beef heart as the protein source lies in the fact that database of bovine proteins is far from being complete compared to those for human and mouse databases. Variations in amino acid sequence of less conserved proteins may potentially preclude successful identification by peptide mass mapping, even if cross-species protein databases are used for MS-Fit search. In most cases, this problem can be overcome by further tandem MS/MS analysis on the individual peptides followed by either EST database search or BLAST (homology database search) on the partial sequences derived from “de novo” peptide sequencing or manual data interpretation. We have used these strategies to identify 56 of polyA- or polyU-binding proteins directly from the NCBI protein database as shown in Table 1. The protein identification has shown high sequence coverage and mass accuracy of better than 10 ppm achieved on the MALDI QqTOF mass spectrometer. Subsequently, the selected peptide sequences were confirmed by MS/MS measurements. MS-Fit search did not provide any reasonable hits for the eight proteins in Table 2 but sufficient homology was retrieved by BLAST
following Bos taurus EST database search on the MS/MS spectra of individual peptides. Identified Proteins with Affinity to PolyA- or PolyUSepharose. Tables 1 and 2 present a nonredundant list of identified polypeptides. Most of the proteins were binding the RNA moiety of polyA/polyU-Sepharose although some nonspecificity caused by protein association with Sepharose matrix cannot be excluded. The majority of the listed proteins belong to the family of oxidoreductases, which utilize NAD+, NADP+, or FAD as a cofactor. Also a significant number of ATP-binding and Coenzyme A dependent enzymes were detected (Figure 1). It is obvious that the affinity to the adenosine moiety present in all of the above cofactors defines polyA-binding of all these proteins. Some of them also bound polyU. Six of the listed proteins have been reported to have specific RNA-binding properties. Binding of high abundance mitochondrial oxidoreductases and other nonspecific proteins to polyAor polyU-Sepharose was not a limiting factor since sample depletion of these proteins prior to chromatography did not result in detection of additional RNA-binding proteins. The fact that we have not detected other known mitochondrial RNAbinding proteins can be explained by the following factors: (1) some proteins are binding only specific RNA sequences or tertiary structures (as is the case of tRNA synthetases, ribosomal proteins and PPR motif containing proteins), (2) relatively low sensitivity of 2-D gel analysis and the need for even larger amounts of starting material, or (3) the need for alternative method of mitochondrial protein extraction. It must be noted that apparently the true number of mitochondrial RNA-binding proteins is not high. The only other proteins reported in proteomic studies were tRNA synthetases, ribosomal proteins, LRPPRC and ribonuclease H1.2,4 Both studies did not detect mitochondrial RNA polymerase or RNAse P. Up to 10 RNA binding proteins were seen in UV-cross-linking experiments to a [32P] labeled transcript of bovine COII mRNA with only one of them positively identified.6 The binding of eight structural nonmitochondrial proteins (#1-6, 57, 58) to RNA-Sepharose was apparently unspecific and their presence in our mitochondrial preparation was just another indication of the tight association between mitochonJournal of Proteome Research • Vol. 4, No. 1, 2005 47
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Figure 1. 2-D gel analysis of proteins eluted from a polyASepharose. (A) Soluble proteins extracted from mitochondria by repeated homogenization in hypotonic buffer and sonication in the presence of 2 mM Mg2+. (B) Proteins from Triton X-100 extract of mitochondrial membranes depleted of abundant soluble proteins in the presence of 10 mM Mg2+. Proteins numbered as in Tables 1 and 2.
dria and cytoskeleton. Some of these proteins were also identified in published mitochondrial proteomics studies.2,4 GAPDH and aldolase are cytosolic enzymes from glycolytic pathway, which consistently show up in mitochondrial proteomic studies2,4 indicating their apparent association with mitochondria. Along with affinity to adenosine, NAD+-dependent GAPDH may possess specific affinity to RNA as it was shown to interact with cis-acting RNAs of human parainfluenza virus type 3 in complex with La protein in vitro and in vivo.9 Among six identified tricarboxylic acid cycle enzymes, two proteins, glutamate dehydrogenase and aconitase, are known for their specific RNA-binding properties. Glutamate dehydrogenase binds a number of mRNAs including those encoding tissue-specific isoforms for cytochrome oxidase subunits.10,11 Aconitase has a cytoplasmic homologue, IRP-1, which in its Fe-S cluster-free form, apoIRP-1, binds to RNA.12 Dihydrolipoamide succinyltransferase, a subunit of oxoglutarate dehy48
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drogenase identified in this study, was also recently found to be bifunctional. A 30 kDa truncated version of the protein, transcribed starting from intron 7 in DSLT gene and localized to mitochondrial intermembrane space, is thought to be involved in the assembly of cytochrome c oxidase or other respiratory complexes.13 Most of the eleven detected mitochondrial polypeptides involved in fatty acid β-oxidation reactions (#42-49, 60, 63, 64) bind Coenzyme A adducts, which accounts for their affinity to polyA. A number of polypeptides from mitochondrial inner membrane respiratory complexes were identified: 8 subunits of Complex I, 2 subunits of Complex II, 3 subunits of Complex III, and 4 subunits of Complex V. Apparently, presence of these polypeptides in the soluble fraction of mitochondrial extract was the result of sonication. Tentative RNA-binding properties of Complex III core subunits are undocumented and quite surprising. AuH protein (RNA-binding homologue of Coenzyme A hydratase), highly expressed in kidney, skeletal muscle, heart, liver and spleen, has been characterized as mitochondrial by immunoelectron microscopy.14 It is bifunctional, exhibiting an AU-specific RNA-binding property and an enoyl-CoA hydratase activity and is thought to link mRNA decay to metabolic reactions.14 Splicing factor 2 (SF2)- associated p32 protein is a conserved eukaryotic mitochondrial matrix protein15, which is apparently present in the cytoplasm and nucleus simultaneously.16 Although its mitochondrial function is obscure it may have a role in regulating constitutive splicing and alternative RNA splicing in the nucleus as it was shown to inhibit RNA-binding by ASF/ SF2, a member of the SR family of splicing factors.17 Estradiol 17 β-dehydrogenase VIII is an enzyme involved in regulation of biologically active estrogens and androgens by inactivating estradiol, testosterone and dihydrotestosterone.18 MITOPRED gives 92.3% confidence value that its mitochondrial and MITOP probability of its import into mitochondria is 0.87. This protein was also reported in the mouse mitochondrial proteomics study4. However, our microscopy data for the expressed GFP-tagged estradiol 17 β-dehydrogenase VIII shows exclusive cytoplasmic localization of the protein (Figure 3, G) and does not support the conclusion that it is mitochondrial protein. Other known mitochondrial identified proteins were electrontransfer flavoprotein-ubiquinone oxidoreductase, creatine kinase, calsequestrin (Ca2+ binding protein), voltage dependent anion channel 1 (porin in the mitochondrial outer membrane), adenylate kinase-3 (AK3) and apoptosis-inducing factor (AIF). Apoptosis-inducing factor (AIF) is a phylogenetically conserved mitochondrial flavoprotein that has both NADH diaphorase and apoptosis inducing activity.19 Protein Isoforms. 2-D electrophoresis and MALDI peptide mass fingerprinting revealed the presence of several isoforms for some identified proteins. Number of apparent isoforms (in parentheses) was as follows (protein numbering as in Tables 1,2): 7(3), 11(>3), 15(2), 18(4), 19(2), 21(5), 22(2), 23(2), 31(>2), 34(2), 35(2), 38(2), 44(3), 46(2), 50(3), 52(2), 56(2), 60(2), 64(>3). Although known precautions were taken to minimize urea reactivity, the possibility that some of the visible isoforms are the result of carbamylation reactions should not be excluded. Except for the PDH E1-R and GAPDH the existence of the isoforms characterized by different pI values for these proteins has not been reported before in a literature. PDH E1-R is known
Proteomics of Mitochondrial RNA-Binding Proteins
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Figure 2. MS/MS spectra of the parent peptide ions (A) at m/z 1369.64 and (B) at m/z 1856.96. Peptide sequences deduced from the MS/MS fragmentations along with the C-terminal y-ions and N-terminal b-ions are shown. The notation used for identifying peptide fragments is based on that proposed by Roepstorff and Fohlman34 as later modified by Biemann35. C*- cystein carbamidomethylation. In the case of the peptide ion at m/z 1369.644, its measured mass is 57.023 Da higher than the theoretical value (MH+, 1312.621 Da) calculated from the sequence (NLSTFAVDGGTCK). The MS/MS fragments of yn ions (n ) 2-12) in Figure 2A defined eleven amino acid residues near the C-terminus according to the mass difference between adjacent peaks. The 57.023 Da increment was thus localized to the two residues (CK) near the N-terminus, suggesting a carbamidomethylation at the cystein introduced by iodoacetamide treatment.
to be phosphorylated in vivo having 3 phosphorylation sites.20 Three specific phosphorylated forms of GAPDH with the pI range in 7.6 to 8.3 were reported to associate with RNAs of human parainfluenza virus type 3.21 Binding Specificity, Relative Abundance and Membrane Association of Selected Proteins. The primary goal of the present study was to identify novel mitochondrial proteins. However, based on performed chromatography experiments, observations can be made for selected identified proteins in relation to (1) column binding specificity (polyA vs polyU affinity and the effect of Mg2+ concentration), (2) relative abundance and (3) mitochondrial membrane association. Most of the identified mitochondrial proteins have been excluded from the analysis, as their RNA-binding is apparently not RNA specific. RNA-binding properties of the TCA cycle enzymes, glutamate dehydrogenase and aconitase, are well-known. In the present study, glutamate dehydrogenase bound polyA at low (2mM) Mg2+ concentration and did not bind polyU. Aconitase bound polyU at high Mg2+ (10 mM) concentration but not polyA. Glycolytic enzymes GAPDH and aldolase bound polyA and polyU respectively at low Mg2+ concentration. Creatine kinase, electron transfer flavoprotein, mitochondrial apoptosis inducing factor and voltage dependent anion channel 1 were binding both polyA and polyU only at high Mg2+ (10 mM). Mitochon-
drial adenylate kinase 3, AuH protein (RNA binding homologue of Coenzyme A hydratase) and carnitine acetyltransferase bound polyA but not polyU at low Mg2+ concentration. Complex III core subunits 1 and 2, surprisingly showed affinity for RNA by binding to polyA and polyU columns respectively at high Mg2+ concentration. Cardiac calsequestrin and SF2p32 pre-mRNA splicing factor bound polyU but not polyA at low Mg2+ concentration. HES1/KNP-I protein extracted with Triton X-100 from mitochondrial membranes previously depleted of abundant soluble proteins was found to bind the polyA column at high Mg2+ concentration. Markedly enriched proteins (the five times higher spot intensity than the average protein spot intensity on the gel) on polyA column included Complex V subunits R, β, and δ; acetyl-Coenzyme A C-acetyltransferase/thiolase; E3 and E1-β of PDH; and ζ-crystallin. Complex V subunit β, PDH E1-β, isocitrate dehydrogenase (NADP+ dependent), dehydrogenase/ 3-ketoacyl-coAthiolase/enoyl-coAhydratase (β subunit) were relatively enriched on the polyU column. ζ-Crystallin/NADPH:Quinone Oxidoreductase is a Cytosolic Protein Associated with Mitochondria. ζ-Crystallin was one of the strikingly enriched soluble proteins on polyA-Sepharose, binding RNA at low Mg2+ concentration (Figure 1A). It has also been detected in the polyA bound fraction of Triton X-100 mitochondrial extract depleted of abundant soluble proteins Journal of Proteome Research • Vol. 4, No. 1, 2005 49
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Ponamarev et al.
Figure 3. Localization of GFP-tagged HES1/KNP1, ζ-crystallin and estradiol 17 β-dehydrogenase 8 within the cell. Human transformed fibroblasts transfected with HES1/KPN1-GFP were incubated with Mitotracker Red for 20 min, fixed and imaged by confocal microscopy. (A) GFP fluorescence. (B) Mitotracker Red. (C) Overlay of A and B. COS-1 cells transfected with HES1/KPN1-GFP and imaged by RTM-3 show green fluorescence pattern (D) exactly corresponding to the mitochondrial network of the same cell visualized in the high contrast mode (E). (F) COS-1 cells transfected with (ζ-crystallin)-GFP and imaged by RTM-3 show cytoplasmic localization of the protein. (G) COS-1 cells transfected with (estradiol 17 β-dehydrogenase)-GFP and imaged by RTM-3 show cytoplasmic localization of the protein.
(Figure 1B), suggesting its mitochondrial membrane association. It is present in the ocular tissues of all vertebrates22,23 and at enzymatic levels in nonlenticular tissues. MITOPRED gives 84.6% confidence that it is mitochondrial, while MITOP24 probability of import into mitochondria is calculated as 0.18 and PSORT (http://psort.nibb.ac.jp) predicts it to be cytosolic. Since ζ-crystallin has been detected in previous comprehensive mitochondrial proteomic study,2 we decided to obtain unambiguous evidence of its intracellular localization by expressing a ζ-crystallin C-terminally fused with GFP in the COS-1 cells. Figure 3 (F) demonstrates that (ζ50
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crystallin)-GFP is present, diffused in the cytosol with no real sign of mitochondrial localization. The nucleic acid binding properties of ζ-crystallin are well documented. It binds different forms of DNA and was speculated to have a role in DNA maintenance.25 A more specific role for ζ-crystallin was determined when it was identified as a protein binding to the pH response elements of mitochondrial glutaminase and glutamate dehydrogenase mRNAs. It specifically binds a direct repeat of an 8-base AU-rich element in 3′-UTR of glutaminase mRNA and a similar sequence in 3′UTR of glutamate dehydrogenase mRNA facilitating pH-
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Proteomics of Mitochondrial RNA-Binding Proteins
responsive stabilization of both mRNAs.26,27 Interestingly, in yeast, glutamate dehydrogenase (yeast gene YDL215C) mRNA and other mRNAs coding for proteins of prokaryotic origin were shown to be translated from mitochondrion-associated polysomes.28,29 This could be a possible mechanism for ζ-crystallin recruitment to the outer mitochondrial membrane. HES1/KNP-I, a Protein of Unknown Function, Resides in Mitochondria. 2-D gel electrophoresis of polyA-bound proteins from Triton X-100 extract of mitochondrial membranes previously depleted of abundant soluble proteins (see Experimental Section) revealed a prominent band with apparent molecular weight 25-30 kDa and pI 8-9, which was identified as HES1/ KNP-I (Figure 1B). The apparent molecular weight and pI values observed on the 2-D gel correlated well with the values calculated for human homologue (28 kDa, pI 8.5). Since peptide mass fingerprinting did not provide a good database hit, positive identification was achieved through MS/MS peptide sequencing of two high-intensity MS peaks at m/z 1369.644 and 1856.956 followed by bovine EST database search (Figure 2). A BLAST search indicated that the peptides NLSTFAVDGGTC*K (C*: cysteine carbamidomethylation) and LTAVNHDAAIFPGGFGAAK are highly homologous to the peptides 142-154(NLSTFAVDGKDCK) and 117-141 (ITDLANLSAANHDAAIFPG-GFGAAK) from the database tryptic digest of human HES1/KNP-I (GenBank accession CAA68857). This protein has been identified in a comprehensive mouse mitochondrial proteomics study under the name “DNA segment, Chr 10, John Hopkins University 81 expressed”.4 HES1 gene (alternative names KNP-I, GT335) characterization came about as a part of the search for candidate genes that may be involved in pathogenesis of several serious genetic diseases mapped to the 21q22.3 locus.30-32 In human chromosome 21 the gene is located between heat shock transcription factor 2 binding protein and ORF29. This protein is highly expressed in heart and skeletal muscle, to a much lesser extent in liver, kidney, pancreas, brain and placenta, and is undetectable in lung.30,32 HES1/KNP-I is 268 aa and 266 aa long in humans and mouse respectively, with 89% identity. MITOPRED predicts it to be mitochondrial with 99% confidence, MITOP probability of import into the mitochondria is 0.96 and PSORT localizes HES1/KNP-I to mitochondria. MitoProteome (http:// www.mitoproteome.org) lists this protein with the data source window left blank. NCBI OMIM annotation for HES1/KNP-I is confused with annotation of another gene with the same name HES1 (Hairy/enhancer of split, gene map locus 3q28-q29), which should be corrected. There are two described homologues - ES1 protein (270 amino acids) in zebrafish33 and SCRP-27A protein in E.coli, with no homologous protein in yeast. No HES1/KNP-I-related genes were found in human or mouse genomes. So far no specific function has been postulated for HES1/KNP-I or homologous proteins. To unequivocally prove mitochondrial localization, HES1/ KNP-I fused C-terminally with GFP was expressed in human fibroblasts and COS-1 cells (see Experimental procedures). Figure 3 (A-C) shows the green fluorescence pattern in human fibroblasts, co-incident with the image of a mitochondrial network visualized with mitotracker red. Images of COS-1 cells expressing HES1/KNP-I-EGFP show even more clear exclusive mitochondrial localization of this protein (Figure 3 (D, E)). Identification of HES1/KNP-I as mitochondrial should stimulate the functional characterization of this protein in the light of its potential involvement in several genetic disorders.30
Acknowledgment. This research was supported by Genome Canada (Ontario Genome Initiative). We are thankful to Richardson Technology Inc. for making the RTM-3 microscope available for this study and Sandy Raha and Alexandra Mohr for assistance in imaging. Ana Florescu, Thomas Hurd, Mary Maj, and Anita Weadge assisted in preparing beef heart mitochondria, which was greatly appreciated. References (1) Sickmann, A.; Reinders, J.; Wagner, Y.; Joppich, C.; Zahedi, R.; Meyer, H. E.; Schonfisch, B.; Perschil, I.; Chacinska, A.; Guiard, B.; Rehling, P.; Pfanner, N.Meisinger, C. The proteome of Saccharomyces cerevisiae mitochondria. Proc. Natl. Acad. Sci. U. S. A. 2003, 100, 13207-13212. (2) Taylor, S. W.; Fahy, E.; Zhang, B.; Glenn, G. M.; Warnock, D. E.; Wiley: S.; Murphy, A. N.; Gaucher, S. P.; Capaldi, R. A.; Gibson, B. W.Ghosh, S. S. Characterization of the human heart mitochondrial proteome. Nat. Biotechnol. 2003, 21, 281-286. (3) Cotter, D.; Guda, P.; Fahy, E.; Subramaniam, S. MitoProteome: mitochondrial protein sequence database and annotation system. Nucleic Acids Res. 2004, 32 Database issue, D463-D467. (4) Mootha, V. K.; Bunkenborg, J.; Olsen, J. V.; Hjerrild, M.; Wisniewski, J. R.; Stahl, E.; Bolouri, M. S.; Ray, H. N.; Sihag, S.; Kamal, M.; Patterson, N.; Lander, E. S.; Mann, M. Integrated analysis of protein composition, tissue diversity, and gene regulation in mouse mitochondria. Cell 2003, 115, 629-640. (5) Guda, C.; Fahy, E.; Subramaniam, S. MITOPRED: a genome-scale method for prediction of nucleus-encoded mitochondrial proteins. Bioinformatics 2004. (6) Koc, E. C.; Spremulli, L. L. RNA-binding proteins of mammalian mitochondria. Mitochondrion 2003, 2, 277-291. (7) Milli, S.; Pinol-Roma, S. LRP130, a pentatricopeptide motif protein with a noncanonical RNA binding domain, is bound in vivo to mitochondrial and nuclear RNAs. Mol. Cell. Biol. 2003, 14, 49724982. (8) Raha, S.; Myint, A. T.; Johnstone, L.; Robinson, B. H. Control of oxygen free radical formation from mitochondrial complex I: roles for protein kinase A and pyruvate dehydrogenase kinase. Free Radic. Biol. Med. 2002, 32, 421-430. (9) De, B. P.; Gupta, S.; Zhao, H.; Drazba, J. A.; Banerjee, A. K. Specific interaction in vitro and in vivo of glyceraldehyde-3-phosphate dehydrogenase and LA protein with cis-acting RNAs of human parainfluenza virus type 3. J. Biol. Chem. 1996, 271, 24728-24735. (10) Preiss, T.; Hall, A. G.; Lightowlers, R. N. Identification of bovine glutamate dehydrogenase as an RNA-binding protein. J. Biol. Chem. 1993, 268, 24523-24526. (11) Preiss, T.; Sang, A. E.; Chrzanowska-Lightowlers, Z. M.; Lightowlers, R. N. The mRNA-binding protein COLBP is glutamate dehydrogenase. FEBS Lett. 1995, 367, 291-296. (12) Gray, N. K.; Hentze, M. W. Iron regulatory protein prevents binding of the 43S translation preinitiation complex to ferritin and eALAS mRNAs. Embo J. 1994, 13, 3882-3891. (13) Kanamori, T.; Nishimaki, K.; Asoh, S.; Ishibashi, Y.; Takata, I.; Kuwabara, T.; Taira, K.; Yamaguchi, H.; Sugihara, S.; Yamazaki, T.; Ihara, Y.; Nakano, K.; Matuda, S.Ohta, S. Truncated product of the bifunctional DLST gene involved in biogenesis of the respiratory chain. Embo J. 2003, 22, 2913-2923. (14) Brennan, L. E.; Nakagawa, J.; Egger, D.; Bienz, K.; Moroni, C. Characterisation and mitochondrial localisation of AUH, an AUspecific RNA-binding enoyl-CoA hydratase. Gene 1999, 228, 8591. (15) Muta, T.; Kang, D.; Kitajima, S.; Fujiwara, T.; Hamasaki, N. p32 protein, a splicing factor 2-associated protein, is localized in mitochondrial matrix and is functionally important in maintaining oxidative phosphorylation. J. Biol. Chem. 1997, 272, 2436324370. (16) Luo, Y.; Yu, H.; Peterlin, B. M. Cellular protein modulates effects of human immunodeficiency virus type 1 Rev. J. Virol. 1994, 68, 3850-3856. (17) Petersen-Mahrt, S. K.; Estmer, C.; Ohrmalm, C.; Matthews, D. A.; Russell, W. C.Akusjarvi, G. The splicing factor-associated protein, p32, regulates RNA splicing by inhibiting ASF/SF2 RNA binding and phosphorylation. Embo J. 1999, 18, 1014-1024. (18) Fomitcheva, J.; Baker, M. E.; Anderson, E.; Lee, G. Y.Aziz, N. Characterization of Ke 6, a new 17β-hydroxysteroid dehydrogenase, and its expression in gonadal tissues. J. Biol. Chem. 1998, 273, 22664-22671.
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Ponamarev et al. (28) Marc, P.; Margeot, A.; Devaux, F.; Blugeon, C.; Corral-Debrinski, M.; Jacq, C. Genome-wide analysis of mRNAs targeted to yeast mitochondria. EMBO Rep. 2002, 3, 159-164. (29) Sylvestre, J.; Vialette, S.; Corral Debrinski, M.; Jacq, C. Long mRNAs coding for yeast mitochondrial proteins of prokaryotic origin preferentially localize to the vicinity of mitochondria. Genome Biol. 2003, 4, R44. (30) Lafreniere, R. G.; Rochefort, D. L.; Kibar, Z.; Fon, E. A.; Han, F.; Cochius, J.; Kang, X.; Baird, S.; Korneluk, R. G.; Andermann, E.; Rommens, J. M.Rouleau, G. A. Isolation and characterization of GT335, a novel human gene conserved in Escherichia coli and mapping to 21q22.3. Genomics 1996, 38, 264-272. (31) Nagamine, K.; Kudoh, J.; Minoshima, S.; Kawasaki, K.; Asakawa, S.; Ito, F.; Shimizu, N. Isolation of cDNA for a novel human protein KNP-I that is homologous to the E. coli SCRP-27A protein from the autoimmune polyglandular disease type I (APECED) region of chromosome 21q22.3. Biochem. Biophys. Res. Commun. 1996, 225, 608-616. (32) Scott, H. S.; Chen, H.; Rossier, C.; Lalioti, M.; D.; Antonarakis, S. E. Isolation of a human gene (HES1) with homology to an Escherichia coli and a zebrafish protein that maps to chromosome 21q22.3. Hum. Genet. 1997, 99, 616-623. (33) Chang, H.; Gilbert, W. A novel zebrafish gene expressed specifically in the photoreceptor cells of the retina. Biochem. Biophys. Res. Commun. 1997, 237, 84-89. (34) Roepstorff, P.; Fohlman, J. Proposal for a common nomenclature for sequence ions in mass spectra of peptides. Biomed. Mass Spectrom. 1984, 11, 601. (35) Biemann, K. Contributions of mass spectrometry to peptide and protein structure. Biomed. Enviro. Mass Spectrom. 1988, 16, 99-111.
PR049872G
