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Decoding Cytochrome C Oxidase Biogenesis: New Insights Into Copper Trafficking

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Decoding Cytochrome C Oxidase Biogenesis: New Insights Into Copper Trafficking University of Pennsylvania ScholarlyCommons Publicly Accessible Penn Dissertations 2011 Decoding Cytochrome C Oxidase Biogenesis: New Insights Into Copper Trafficking Nursel Seda Ekici University of Pennsylvania, [email protected] Follow this and additional works at: https://repository.upenn.edu/edissertations Part of the Biology Commons, Genetics Commons, and the Molecular Biology Commons Recommended Citation Ekici, Nursel Seda, "Decoding Cytochrome C Oxidase Biogenesis: New Insights Into Copper Trafficking" (2011). Publicly Accessible Penn Dissertations. 474. https://repository.upenn.edu/edissertations/474 This paper is posted at ScholarlyCommons. https://repository.upenn.edu/edissertations/474 For more information, please contact [email protected]. Decoding Cytochrome C Oxidase Biogenesis: New Insights Into Copper Trafficking Abstract Acquisition, delivery and incorporation of metals to their respective metalloproteins are important cellular processes. These processes are tightly controlled so that cells are not exposed to free metal concentrations that would lead to harmful oxidative damages. Cytochrome c oxidases (Cox) are among these metalloproteins whose assembly and activity involves incorporation of Cu cofactor into their catalytic subunits in addition to the maturation of other subunits. In this study, we focused on the pathways of acquisition of Cu by the facultative phototroph Rhodobacter capsulatus for incorporation into the heme–Cu binuclear center of its cbb3–type Cox (cbb3–Cox). Genetic screens identified a 2+ cbb3–Cox defective mutant that requires Cu supplement to produce an active cbb3–Cox. Complementation of this mutant using wild-type genomic libraries unveiled a novel gene (ccoA) required for cbb3–Cox biogenesis in R. capsulatus. In the absence of CcoA, cellular content of Cu decreases, and cbb3–Cox assembly and activity becomes defective. CcoA shows pronounced homology to Major Facilitator Superfamily (MFS) type transporter proteins. Members of this family are known to transport small solutes or drugs, but so far, no MFS protein was implicated in cbb3–Cox biogenesis. In order to dissect the mechanism of Cu acquisition in the absence of CcoA, we isolated ΔccoA mutants that were cbb3–Cox defective after addition of Cu. Characterization of these mutants by genetic complementations revealed mutations in cytochrome c maturation (CCM) genes. These mutants were able to grow photosynthetically on the contrary to the usual phenotype of CCM genes deletion mutants. Here we show that these mutations are not directly involved in the Cu traffickingo t CcoN but involved in the production of membrane bound cytochrome c subunits of cbb3–Cox. Although this study provides additional information about CCM system in R. capsulatus, the additional pathways of Cu acquisition to cbb3–Cox in the presence of exogenous Cu still remains to be identified. In the future, determination of ccoA bypass mutations will provide novel insights on the maturation and assembly of membrane-integral metalloproteins, and on hitherto unknown function(s) of MFS type transporters in bacterial Cu acquisition. Degree Type Dissertation Degree Name Doctor of Philosophy (PhD) Graduate Group Biology First Advisor Fevzi Daldal Keywords Copper trafficking, Cytochrome c oxidase, Metalloproteins, Respiration, Rhodobacter capsulatus Subject Categories Biology | Genetics | Molecular Biology This dissertation is available at ScholarlyCommons: https://repository.upenn.edu/edissertations/474 DECODING CYTOCHROME C OXIDASE BIOGENESIS: NEW INSIGHTS INTO COPPER TRAFFICKING Nursel Seda Ekici A DISSERTATION in Biology Presented to the Faculties of the University of Pennsylvania in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy 2011 Supervisor of Dissertation _______________________________ Fevzi Daldal, Ph.D., Professor of Biology Graduate Group Chairperson _______________________________ Doris Wagner, Ph.D., Associate Professor of Biology Dissertation Committee Andrew Dancis, M.D., Associate Professor of Medicine Mark Goulian, Ph.D., Associate Professor of Biology Mecky Pohlschroder, Ph.D., Associate Professor of Biology Philip A. Rea, Ph.D., Professor of Biology Jun Zhu, Ph.D., Assistant Professor of Microbiology DECODING CYTOCHROME C OXIDASE BIOGENESIS: NEW INSIGHTS INTO COPPER TRAFFICKING COPYRIGHT 2011 Nursel Seda Ekici Dedicated to my family… iii ACKNOWLEDGMENT First, I would like to express my gratitude to my thesis advisor Fevzi Daldal for giving me this opportunity to be one of his students. I learned how to do research in a critical, logical, passionate and controlled manner from him. He is not only an enthusiastic, motivating, critical, challenging mentor but also a family to me. I am indebted to my committee members, Dr. Andy Dancis, Dr. Mark Goulian, Dr. Mecky Pohlschroder, Dr. Phil Rea, and Dr. Jay Zhu for sharing their invaluable insights about my project, for their support, advice and keeping me on track during my PhD. Also I wish to express my sincere thanks to Goulian and Dancis lab members for their help during my rotations. I would like to thank all past and present Daldal lab members that all helped in different aspects of my PhD. I would like to thank Dr. Serdar Turkarslan and Dr. Özlem Önder for teaching me the lab techniques about Rhodobacter capsulatus during my first years, Dr. Nur Selamoglu, Dr. Honghui Yang, Xinpei Jiang, Duygu Soysal, Ibrahim Sektioglu, Xiaomin Wu, Gulgez Gokce Yildiz for contributing to my project and their helpful discussions, Dr. Bahia Khalfaoui Hassani, Dr. Andreia Verissimo, Dr. Pascal Lanciano for critical reading of my thesis, and Dr. Hans-Georg Koch and Grzegorz Pawlik for our fruitful collaboration and discussions. I would like to thank to my family, my mom and dad for raising me and making me who I am today and their unconditional love, to my sister for always cheering me up and my uncles for their endless life strategies and support. iv I would like to thank to all my friends who made this journey possible by sharing my life, listening to my problems and complaints and sometimes by just simply being next to me. Especially thanks to Serdar for always cheering me up, showing me the positive side in everything and be there for me all the time. Thanks to Ezgi, Gokce, and Myat for their support and for the countless great memories and fun we had together through the years. Finally, I would like to thank Rhodobacter capsulatus for making this thesis possible. Without its cooperation, it would not be possible! And I’m sorry for killing billions of you with NADI staining! v ABSTRACT DECODING CYTOCHROME C OXIDASE BIOGENESIS: NEW INSIGHTS INTO COPPER TRAFFICKING Nursel Seda Ekici Dr. Fevzi Daldal Acquisition, delivery and incorporation of metals to their respective metalloproteins are important cellular processes. These processes are tightly controlled so that cells are not exposed to free metal concentrations that would lead to harmful oxidative damages. Cytochrome c oxidases (Cox) are among these metalloproteins whose assembly and activity involves incorporation of Cu cofactor into their catalytic subunits in addition to the maturation of other subunits. In this study, we focused on the pathways of acquisition of Cu by the facultative phototroph Rhodobacter capsulatus for incorporation into the heme-Cu binuclear center of its cbb3-type Cox (cbb3-Cox). Genetic 2+ screens identified a cbb3-Cox defective mutant that requires Cu supplement to produce an active cbb3-Cox. Complementation of this mutant using wild-type genomic libraries unveiled a novel gene (ccoA) required for cbb3-Cox biogenesis in R. capsulatus. In the absence of CcoA, cellular content of Cu decreases, and cbb3-Cox assembly and activity becomes defective. CcoA shows pronounced homology to Major Facilitator Superfamily (MFS) type transporter proteins. Members of this family are known to transport small solutes or drugs, but so far, no MFS protein was implicated in cbb3-Cox biogenesis. In order to dissect the mechanism of Cu acquisition in the absence of CcoA, we isolated vi ΔccoA mutants that were cbb3-Cox defective after addition of Cu. Characterization of these mutants by genetic complementations revealed mutations in cytochrome c maturation (CCM) genes. These mutants were able to grow photosynthetically on the contrary to the usual phenotype of CCM genes deletion mutants. Here we show that these mutations are not directly involved in the Cu trafficking to CcoN but involved in the production of membrane bound cytochrome c subunits of cbb3-Cox. Although this study provides additional information about CCM system in R. capsulatus, the additional pathways of Cu acquisition to cbb3-Cox in the presence of exogenous Cu still remains to be identified. In the future, determination of ccoA bypass mutations will provide novel insights on the maturation and assembly of membrane-integral metalloproteins, and on hitherto unknown function(s) of MFS type transporters in bacterial Cu acquisition. vii TABLE OF CONTENTS COPYRIGHT ................................................................................................................... ii DEDICATION ................................................................................................................ iii ACKNOWLEDGMENT ................................................................................................ iv ABSTRACT .....................................................................................................................vi TABLE OF CONTENTS
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