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Unveiling the Regulatory Network for Di/Tri-Peptide Utilization in Saccharomyces Cerevisiae

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University of Tennessee, Knoxville TRACE: Tennessee Research and Creative Exchange Doctoral Dissertations Graduate School 5-2006 Unveiling the Regulatory Network for Di/tri-peptide Utilization in Saccharomyces cerevisiae Houjian Cai University of Tennessee - Knoxville Follow this and additional works at: https://trace.tennessee.edu/utk_graddiss Part of the Microbiology Commons Recommended Citation Cai, Houjian, "Unveiling the Regulatory Network for Di/tri-peptide Utilization in Saccharomyces cerevisiae. " PhD diss., University of Tennessee, 2006. https://trace.tennessee.edu/utk_graddiss/1649 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 Houjian Cai entitled "Unveiling the Regulatory Network for Di/tri-peptide Utilization in Saccharomyces cerevisiae." 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 Microbiology. Jeffrey M. Becker, Major Professor We have read this dissertation and recommend its acceptance: David A. Brian, Pamela L. C. Small, Todd Reynolds, Bruce McKee 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 Houjian Cai entitled “Unveiling the Regulatory Network for Di/tri-peptide Utilization in Saccharomyces cerevisiae.” 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 Microbiology. Jeffrey M. Becker Major Professor We have read this dissertation and recommend its acceptance: David A. Brian . Pamela L. C. Small . Todd Reynolds . Bruce McKee . Accepted for the Council: Anne Mayhew . Vice Chancellor and Dean of Graduate Studies (Original signatures are on file with official student records.) Unveiling the Regulatory Network for Di/tri-peptide Utilization in Saccharomyces cerevisiae A Dissertation Presented for the Doctor of Philosophy Degree The University of Tennessee, Knoxville Houjian Cai May 2006 DEDICATION My faith was never shaken. It took me 15 years from the time when I planned to earn a Ph.D degree. This work is dedicated to my family: To my passed eldest brother, who mistakenly sacrificed himself to the distorted era of his country when he still was not able to think clearly; whose spirit is my everlasting driving force for life. To my passed grandmother, who raised me up on her back, and her mother-image is in my memory forever. It was greatly unfortunate that I could not finish up my study before she passed away. To my wife, who has supported me and always makes me smarter. To my brothers, Houyong and Houren, and sister, Sujuan for caring about me and taking care of the family when I was away from home in the past years. To my mother, who always worries about my living outside the country and never gave up her faith for my success. Finally, to my daughter, Lena, who brings the joy to me. Her sweet smile makes me energetic whenever I am tired. ii ACKNOWLEDGEMENTS Life is really un-predictable. Fifteen years ago I had never known that I would finish up my Ph.D study in Dr. Becker’s lab. It is my fortune that I have met so many great people in the journey of reaching this degree. The encouragement and support of these people has made possible the realization of my dream, getting a Ph.D degree. It is more than difficult to adequately express my gratitude for their help. I would like to acknowledge Dr. Dittmar Hahn for providing me an opportunity to study in his lab, which brought me from Europe to the US. He showed me how to work efficiently in being a scientist. I would also like to thank Dr. Gary Stacy for recruiting me to the University of Tennessee, Knoxville. The dissertation committee members made it possible for me to go smoothly in my projects. I would like to thank Dr. Pamela Small, Dr. David Brian, Dr. Todd Reynolds, and Dr. Bruce McKee, for being interested in my ideas and making my time as a graduate student the most challenging adventure I have yet undertaken. I would also like to thank Dr. Dong Xu provided me a lot of fresh ideas when I started my project. My appreciation goes out to Dr. Fred Naider for providing me the toxic peptides continually, and giving me his faith for whatever I am working on. If I could attribute any one aspect as having contributed most to my success, it would be the talented and supportive people in Becker’s lab. I can not express more of my gratitude to the “lab mom” Dr. Melinda Hauser. I started my lab rotation with her. It was her charming knowledge that became one of reasons to work in the lab. She has been a dictionary for me throughout the time in the lab on everything. My special thanks also iii go to Dr. Tom Masi, a king of molecular biology in the lab. I could always find answers from him about the experimental difficulties, and he usually is the person to crack the first version of my terrible writing. I owed him for spending a lot of free time on understanding my writing at home. Special thanks to Sarah Kauffman, a super-technician, who conquered the boring and helped me to transfer a plasmid more than two hundred times. Thanks to Dr. Amy Wiles, who taught me a lot of English, showed me American culture, and discussed American political issues. She shared the Transporter niche with me in the lab and provided a lot of valuable ideas. Appreciation to Dr. Yong-Hun Lee, who always challenged me in science with the final conclusion “it is not enough” and “so what”. Thanks to my friend, Dr. Cagdas Son, a king in the protein chemistry and computer knowledge in the lab, who showed me a lot of tricks in working with the protein and solving all kinds of computer problems. I enjoyed his humor and shared a lot of life experience. Thanks to Steve Minkin, who came through many times when I needed a laugh. His enthusiasm to political issues and optimum to the life made me understand more about this country. Thanks to Dr. Byung-Kwon Lee, who gave me a lot of suggestion and advice in the scientific field. Special gratitude to Dr. Agnieszka Janiak for a lot of constructive discussions about my project. Thanks to Heejung Kim, George Umanah, Li-Yin Huang, who showed great passion in the lab. I appreciate the enjoyable moment they have brought to me. To my mentor, teacher, Dr. Jeffrey Becker, more than anyone else, I am very grateful for the opportunity he gave to me to work in his lab. In Chinese, we said “one day being your mentor, forever being your father”. In past five years, Jeff’s influence and lessons have permeated my life and instilled me how to face the challenge and conquer iv difficulties. He provided me with strength, support and direction, and gave enormous freedom, patience, and trust in my research. His critical thinking, self-confidence, and open-mind influence my research in the future. I am proud to declare my thanks. v ABSTRACT Dipeptides and tripeptides serve as important sources of amino acids, nitrogen and carbon for the growth of all organisms. To identify genes involved in the regulation of small peptide utilization, I performed a systematic, functional examination of this process in a haploid, non-essential, single-gene deletion mutant library of Saccharomyces cerevisiae. In addition, we used high-throughput phenotyping in which we grew yeast cells on 284 different dipeptides or 11 tripeptides as the sole nitrogen source to dissect different mechanisms of di/tri-peptide utilization in seven genetically diverse strains. I have identified 103 candidate genes involved in regulating peptide utilization: 57 genes whose deletion decreased dipeptide utilization and 46 genes whose deletion enhanced dipeptide utilization. Since in S. cerevisiae the membrane transport protein Ptr2p, encoded by PTR2, mediates the uptake of di/tri-peptides and is a key player in the di/tri- peptide utilization process, I focused our studies on the regulation of PTR2 expression, Ptr2p-GFP localization, and dipeptide uptake assays. Forty-two genes were ascribed to the regulation of PTR2 expression, 37 genes were involved in Ptr2p localization, and 24 genes apparently did not affect expression or localization. Together with the high- throughput phenotyping study, we have identified gene components involved in regulation of dipeptide utilization in many cellular processes: 1) proteins involved in up- or down-regulation of PTR2 transcription including the chromatin remodeling INO80 protein complex (Arp5p, Arp8p, Ies6p and Taf14p), transcription factors (Cup9p, Rpn4p, Stp2p, and Dal81p), polymerase mediators (Ssn3p/Srb10p, Ssn8p/Srb11p, Ssn2p/Srb9p, and Srb8p), and mRNA maturation (Kem1p and Pat1p); 2) proteins involved in the Ptr2p vi trafficking system including ESCRT I, II, III protein complex; 3) proteins participating in metabolic processes including the pyruvate dehydrogenase complex (Pdx1p, Lpd1, Pdb1p, and Lat1p), the fatty acid synthase complex (Oar1p, Mct1p, and Etr1p), and the glycine decarboxylase multienzyme complex (Gcv2p, Gcv3p, and Lpd1p); 4) proteins involved in other cellular processes, such as Ybt1p, a protein located at the vacuole membrane likely related to the storage of dipeptides in the cytosol and Dal5p previously identified as an allantoate/ureidosuccinate permease, which facilitates di/tri-peptide transport.
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