University of Tennessee, Knoxville TRACE: Tennessee Research and Creative Exchange Doctoral Dissertations Graduate School 5-2007 Bioinformatic and Proteomic Investigation of Chloroplast Transit Peptide Motifs and Genesis David R. McWilliams University of Tennessee - Knoxville Follow this and additional works at: https://trace.tennessee.edu/utk_graddiss Part of the Life Sciences Commons Recommended Citation McWilliams, David R., "Bioinformatic and Proteomic Investigation of Chloroplast Transit Peptide Motifs and Genesis. " PhD diss., University of Tennessee, 2007. https://trace.tennessee.edu/utk_graddiss/243 This Dissertation is brought to you for free and open access by the Graduate School at TRACE: Tennessee Research and Creative Exchange. It has been accepted for inclusion in Doctoral Dissertations by an authorized administrator of TRACE: Tennessee Research and Creative Exchange. For more information, please contact [email protected]. To the Graduate Council: I am submitting herewith a dissertation written by David R. McWilliams entitled "Bioinformatic and Proteomic Investigation of Chloroplast Transit Peptide Motifs and Genesis." I have examined the final electronic copy of this dissertation for form and content and recommend that it be accepted in partial fulfillment of the equirr ements for the degree of Doctor of Philosophy, with a major in Life Sciences. Barry D. Bruce, Major Professor We have read this dissertation and recommend its acceptance: Robert L. Hettich, Igor B. Jouline, Frank W. Larimer, Stevern W. Wilhelm Accepted for the Council: Carolyn R. Hodges Vice Provost and Dean of the Graduate School (Original signatures are on file with official studentecor r ds.) To the Graduate Council: I am submitting herewith a dissertation written by David R. McWilliams entitled “Bioin- formatic and Proteomic Investigation of Chloroplast Transit Peptide Motifs and Genesis”. I have examined the final electronic copy of this dissertation for form and content and recommend that it be accepted in partial fulfillment of the requirements for the degree of Doctor of Philosophy, with a major in Life Sciences. Barry D. Bruce Major Professor We have read this dissertation and recommend its acceptance: Robert L. Hettich Igor B. Jouline Frank W. Larimer Steven W. Wilhelm Accepted for the Council: Linda Painter Interim Dean of Graduate Studies (Original signatures on file with official student records.) Bioinformatic and Proteomic Investigation of Chloroplast Transit Peptide Motifs and Genesis A Dissertation Presented for the Doctor of Philosophy Degree The University of Tennessee, Knoxville David R. McWilliams May 2007 Dedication I dedicate this dissertation to my incomparable wife Susanne Carroll McWilliams With whom any project seems not only possible, but an adventure And to my children Jennifer, Andrew, and Anthony Whose support, encouragement, challenge, and humor helped immeasurably ii Acknowledgments I would like to acknowledge all those who have assisted me in this project. First, I would like to especially thank my advisor, Dr. Barry D. Bruce, for taking a chance with a ’non- traditional’ student, and a not very compliant or humble one at that. I would also like to thank my advisory committee, Drs. Robert Hettich, Igor Jouline, Frank Larrimer and Steve Wilhelm for their time and patience demanding hypothesis-driven instead of data- driven research. I thank Dr. Nathan VerBerkmoes for training me on the LCQ, and Dr. Hayes McDonald for helpful discussion on chromatography and other technique matters. Thanks are also due to Dr. Fu-Min Menn for help in trouble-shooting and exorcising LCQ demons. Finally, I would like to thank the past and present members of the Bruce laboratory: Anton Mitsky, Evan Reddick, Michael Vaughn, and Sarah Wright for trying to teach me biochemistry technique, sample isolation, and general biology lore. iii Abstract The eukaryotic mitochondrion was formed by the endosymbiotic association of an α- proteobacterium and a primordial phagocytic eukaryote. A second, and later, endosym- biosis between the eukaryote and a cyanobacterium gave rise to the chloroplast of plants. Following each of these events most of the organellar DNA was exported to the nucleus. A system evolved wherein proteins produced on cytosolic ribosomes are targeted to or- ganelle protein translocators by N-terminal targeting sequences. Protein sorting between the chloroplast and the mitochondrion in the plant cell by the general import pathways shows remarkable fidelity despite a lack of sequence conservation among transit peptides and pre-sequences and despite very little sequence difference between these two targeting peptides. There is evidence for a hydrophobic recognition motif in mitochondrial pre- sequences, and a similar motif has been proposed for the chloroplast transit peptide. We have developed novel motif-finding methods and applied them to our own chloroplast pro- teome data and to literature mitochondrial data. We fail to find a hydrophobic motif that discriminates the chloroplast and the mitochondrion. Another little understood phe- nomenon of organelle protein trafficking is how the targeting sequence is acquired after transfer of organelle DNA to the nucleus. It has been hypothesized that the transit pep- tide is acquired by exon shuffling. We find no correlation of transit peptide lengths with exon boundaries. Furthermore, using highly expressed cyanobacterial proteins conserved in plants, we find that the transit peptide appears as likely to be attached within the primor- dial sequence as without, indicating a more stochastic process for the origin of the transit peptide. iv Contents Introduction 1 1 Comparison of MS/MS Search Algorithms 16 1.1 Abstract..................................... 16 1.2 Introduction................................... 16 1.3 MaterialsandMethods............................. 18 1.4 Results...................................... 18 1.5 Discussion.................................... 29 2 Chloroplast Proteomics 31 2.1 Abstract..................................... 31 2.2 Introduction................................... 31 2.3 MaterialsandMethods............................. 34 2.3.1 Plantproduction ............................ 34 2.3.2 Organelleisolation ........................... 34 2.3.3 Massspectrometry ........................... 35 2.3.4 Massspectrometricdataanalysis . .. 36 2.3.5 Bioinformatics.............................. 36 2.4 Results...................................... 37 2.4.1 MSSearchStrategyComparison. 38 2.4.2 Localizationprediction . 41 2.4.3 ConservationinCyanobacteria. .. 46 2.4.4 Functionalcharacterization . .. 49 2.5 Discussion.................................... 50 3 Motif Analysis of Chloroplast Transit Peptides 54 3.1 Abstract..................................... 54 3.2 Introduction................................... 54 3.3 MaterialsandMethods............................. 59 3.4 Results...................................... 60 3.4.1 Application of MEME to targeting sequences . .... 60 3.4.2 Heuristic analysis of targeting sequences . ...... 63 3.5 Discussion.................................... 77 v 4 Synechocystis PCC 6803 Proteomics 79 4.1 Abstract..................................... 79 4.2 Introduction................................... 79 4.3 MaterialsandMethods............................. 81 4.3.1 Synechocystis cultureandfractionation . 81 4.3.2 Proteindigestion ............................ 81 4.3.3 Column packing and sample loading . 81 4.3.4 High performance liquid chromatography . .... 82 4.3.5 Massspectrometryconditions . 82 4.3.6 Massspectrometricdataanalysis . .. 83 4.3.7 Bioinformaticanalyses . 84 4.4 Results...................................... 85 4.4.1 TheExperimentalProteome . 85 4.4.2 Protein Detection Frequency is Proportional to Codon Adaptation Index................................... 91 4.4.3 Experimental proteins are more likely to be conserved in plants . 93 4.5 Discussion.................................... 97 4.5.1 Proteomics as expression analysis . ... 97 4.5.2 Conservationinplants . .. .. 98 5 Stochastic Nature of Transit Peptide Acquisition 100 5.1 Abstract..................................... 100 5.2 Introduction................................... 100 5.3 MaterialsandMethods............................. 101 5.3.1 Comparison of exon and targeting sequence length . ...... 101 5.3.2 Determination of transit peptide attachment points . ........ 102 5.3.3 Calculation of random targeting sequences . ..... 104 5.4 Results...................................... 104 5.4.1 Correlation of targeting sequence length and exon boundaries . 104 5.4.2 Distribution of predicted transit peptide ‘attachmentpoints’ . 107 5.4.3 Predicted targeting specificity of random sequence . ........ 113 5.5 Discussion.................................... 116 Summary 120 References 124 Appendix 139 A Chloroplast Proteome Results - Sequest 140 B Chloroplast Proteome Results - Three Algorithms 148 C Top-Hits to Proteobacteria 159 vi D Top-Hits to Yeast 161 E Synechocystis Proteins 164 Vita 174 vii List of Tables 1.1 Pearson Correlation Coefficients for Search Scoresa ............. 19 1.2 Estimate of Single-Algorithm False Rejection Ratea ............. 23 2.1 Novel localization prediction algorithms cited in PubMed........... 33 2.2 Sequest Two Peptide Protein Identificationsa ................. 39 2.3 Combined Algorithm Predictiona ....................... 39 2.4 TMHMM Predictions for the Two Search Strategies . ...... 39 2.5 Proteins Identified by ‘Any2’ Not Represented in ‘Seq2’ Results ...... 42 2.6 Localization