Evolution of the eukaryotic membrane trafficking system as revealed by comparative genomic and phylogenetic analysis of adaptin, golgin, and SNARE proteins by Lael Dan Barlow A thesis submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Physiology, Cell, and Developmental Biology Department of Biological Sciences University of Alberta c Lael Dan Barlow, 2019 Abstract All eukaryotic cells possess a complex system of endomembranes that functions in traffick- ing molecular cargo within the cell, which is not observed in prokaryotic cells. This membrane trafficking system is fundamental to the cellular physiology of extant eukaryotes, and includes or- ganelles such as the endoplasmic reticulum, Golgi apparatus, and endosomes as well as the plasma membrane. The evolutionary history of this system offers an over-arching framework for research on membrane trafficking in the field of cell biology. However, the evolutionary origins of this system in the evolution from a prokaryotic ancestor to the most recent common ancestor of extant eukaryotes is a major evolutionary transition that remains poorly understood. A leading paradigm is described by the previously proposed Organelle Paralogy Hypothesis, which posits that coordi- nated duplication and divergence of genes encoding organelle-specific membrane trafficking pro- teins underlies a corresponding evolutionary history of organelle differentiation that produced the complex sets of membrane trafficking organelles found in extant eukaryotes. This thesis focuses on investigating the evolution of families of proteins that sustain membrane traffic by organizing vesicle transport between specific compartments. Comparative genomics and phylogenetic analysis methods were applied to trace the evolution of subunits of the Adaptor Protein complexes, which function in vesicle formation, revealing that losses of genes encoding these subunits in Fungi and duplications in plants are relatively recent events, consistent with a highly conserved role of members of this protein family in organizing the membrane trafficking system. Similar methods were applied to the golgin proteins, which extend from membranes of the Golgi apparatus to recognize and tether specific vesicles. This re- vealed considerable conservation of golgins specific to different regions of the Golgi, consistent with ancestral complexity of the Golgi inferred from conservation of morphological complexity ii among eukaryotes. The Soluble N-ethylmaleimide sensitive factor Attachment protein REceptor (SNARE) protein superfamily comprises numerous proteins forming complexes that mediate vesi- cle fusion at specific locations in the cell. An extensive analysis of the SNAREs revealed evidence for additional SNARE proteins present in the last common ancestor of eukaryotes. Finally, the evo- lution of the four constituent families within the SNARE superfamily was explored and relevance to various potential scenarios of early evolution of the membrane trafficking system are discussed. Taken together, the results of this work shed light on the connections between protein evolu- tion and organelle evolution, and provide novel evidence for distinguishing between alternative scenarios for the evolution of the membrane trafficking system. iii Preface Chapter 2 (exclusive of the preface and afterword) has been published as Barlow, L.D., Dacks, J.B., Wideman, J.G., 2014. From all to (nearly) none: Tracing adaptin evolution in Fungi. Cellu- lar Logistics 4, e28114. https://doi.org/10.4161/cl.28114. This is an open-access article licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported License (https: //creativecommons.org/licenses/by-nc/3.0/). All text in Chapter 2 (exclusive of the pref- ace and afterword) is unmodified from this publication, with the exception of some additional detail added to figure legends. JGW and JBD conceived of and designed the study. JGW performed most of the similarity searches and drafted the manuscript. I performed all phylogenetic analyses, gen- erated figures, and contributed to writing the manuscript. Chapter 3 (exclusive of the preface and afterword) has been published as Larson, R.T., Dacks, J.B., and Barlow, L.D., 2019. Recent gene duplications dominate evolutionary dynamics of adaptor protein complex subunits in embryophytes. Traffic (in press) https://onlinelibrary.wiley. com/doi/abs/10.1111/tra.12698. I obtained permission from John Wiley & Sons, Inc. to re- produce this work in this thesis, and the license number is 4695550381953. All text in Chapter 3 (exclusive of the preface and afterward) is unmodified from this publication (with the exception of a minor correction to the definition of the acronym HSP). With my mentorship, RTL performed similarity searches and phylogenetic analyses for this work as part of one of her undergraduate projects in the Dacks laboratory. JBD initially conceived of the study. RTL and I designed and performed the analyses, and wrote the manuscript. I conceived of and wrote all computer scripts used for performing the analyses. RTL and I prepared figures. Chapter 4 (exclusive of preface and afterword) was published as Barlow, L.D., Nyvltov` a,` E., Aguilar, M., Tachezy, J., Dacks, J.B., 2018. A sophisticated, differentiated Golgi in the ancestor of eukaryotes. BMC Biology 16. https://doi.org/10.1186/s12915-018-0492-9. This article was distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/). All text in Chapter 4 (exclusive of pref- ace and afterword) is unmodified from this publication, with the exception of changes applied as indicated in the published correction to the article (https://rdcu.be/bR35t). EN and JT de- signed, performed and interpreted the experimental work on Mastigamoeba balamuthi, and also iv wrote the relevant manuscript sections. MA and I performed genomic analysis of M. balamuthi. I performed all molecular evolutionary analyses. JBD and I designed the molecular evolutionary analyses, interpreted the results, and wrote the manuscript. Bioinformatic analyses in Chapter 5 are in preparation for eventual submission as a first-author publication with Joel B. Dacks. The idea for this project was initially conceived of by JBD. I per- formed all the work, including conceiving of and writing all computer scripts used for performing the analyses. Though intriguing, experimental work done with Dictyostelium discoideum in Chap- ter 5 is inconclusive and therefore is not currently intended for publication with the phylogenetic results. I performed this experimental work during a 6 week visit to the laboratory of Robert R. Kay at the Medical Research Council Laboratory of Molecular Biology, where I was instructed in experimental methods primarily by Gareth Bloomfield. Therefore, it is important to note that this experimental work was done in collaboration with RRK and GB. v Acknowledgements During my graduate studies I was supported by a Post-Graduate Scholarship-Doctoral (PGS-D) from the National Sciences and Engineering Council of Canada (NSERC), a President’s Doctoral Prize of Distinction and a 75th Anniversary Award from the University of Alberta, a Short Term Travelling Fellowship from the Company of Biologists (Cambridge, England), two Holtz-Connor travel awards from the International Society of Protistologists, and a travel award from the Interna- tional Society for Evolutionary Protistology. Significant funds for covering my stipend also came from NSERC Discovery grants awarded to my supervisor Joel Dacks. Chapter 2 specific acknowledgements: I am greatful for the opportunity to work with my co-authors on the associated publication: Joel B. Dacks and Jeremy G. Wideman. This work was supported by funding awarded to Joel B. Dacks from the following sources: An NSERC Discovery Grant, a New Faculty Award from Alberta Innovates Technology Futures, and the Canada Research Chairs program. We (myself and the other authors of the associated publication) would like to thank the members of the Dacks lab for discussion, as well as K. James, B. Kopetski, C. Lavergne, S. McCormack, N. Ellis, and C. Thulin for organizational and administrative support. Chapter 3 specific acknowledgements: It was an honour to work with my co-authors Raegan T. Larson and Joel B. Dacks on the associated publication. This project could not have happened without Raegan’s hard work and enthusiasm for plants. This work was supported by (NSERC) Discovery grants (RES0021028, RES0043758, RES0046091) and Joel B. Dacks is the Canada Research Chair (Tier II) in Evolutionary Cell Biology. Raegan T. Larson was supported by an Office of the Provost and Vice President (Academic) Summer Student Award from the University of Alberta Faculty of Medicine and Dentistry. For this work, I was supported by my NSERC PGS-D award. Chapter 4 specific acknowledgements: I very much appreciate the important contributions of my co-authors on the associated publication: Eva Nyvltov` a,` Maria Aguilar, Jan Tachezy, and Joel B. Dacks. In particular, Eva and Jan provided all the Mastigamoeba balamuthi data including ge- nomic data and localization experiments. We (myself and co-authors on the associated publication) would like to thank Christen M. Klinger for collaboration on informatics workflows used for run- ning homology searches and all members of the Dacks lab, past and present, for helpful discussion. We also want to thank Drs Aaron Turkewitz, Paul Melancon, Alan Warren and Frances Brodsky for helpful discussion. In performing