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dc.contributor.advisorMonk, Brian
dc.contributor.advisorCannon, Richard
dc.contributor.advisorNiimi, Kyoko
dc.contributor.authorYellagunda Ramalinge Gowda, Sujan
dc.date.available2014-10-28T03:03:46Z
dc.date.copyright2014
dc.identifier.citationYellagunda Ramalinge Gowda, S. (2014). Heterologous expression of membrane proteins in Saccharomyces cerevisiae (Thesis, Doctor of Philosophy). University of Otago. Retrieved from http://hdl.handle.net/10523/5075en
dc.identifier.urihttp://hdl.handle.net/10523/5075
dc.description.abstractThe ATP-binding cassette (ABC) transporters superfamily is one of the largest integral membrane protein families. These multi-domain proteins are ubiquitiously present in all biological kingdoms. To date, 51 ABC genes have been identified in human genome. Of these, predominantly, overexpression of ABCB1, ABCC1 and ABCG2 is associated with multidrug resistance in tumour cells. ABCG2 is a half transporter with reverse topology, that has a nucleotide binding domain that precedes the transmembrane domain (NBD-TMS6), as compared to ABCB1 and ABCC1 which are full transporters with (TMS6-NBD)2 topology. This unique topological feature of ABCG2 is similar to fungal PDR transporters, in particular to proteins that belong to cluster F. ABC transporters are expressed in miniscule amounts in native tumour cells or in other mammalian expression system. Hence, expression of these proteins in a heterologous host is imperative for biomedical research and for the pharmaceutical industry. Despite the current advanced state of heterologous protein expression technology, preparation of high-quality functional protein samples in sufficient amounts by in vitro approaches is still a major bottleneck for the characterization and structural studies of membrane proteins. Therefore, in this thesis work, the possibility of developing Saccharomyces cerevisiae AD∆ strain as a host model expression system for heterologous expression of human ABC transporters was investigated. Human ABCG2 was used as a model protein throughout this study because of its unique characteristic features and phylogenetic relevance to orthologous proteins in Saccharomyces cerevisiae. Despite a fungal PDR transporter – like topology and close phylogenetic evolution, expression of ABCG2 in S. cerevisiae has yet to yield significant quantities of functional enzyme as host biology can make heterologous expression and correct localisation of this transporter a challenge. To obtain ABCG2 in functionally folded conformation many promising approaches were investigated. In this regard, several ABCG2 mutants or PDR-ABCG2 chimera proteins were created; their expression, localisation and drug efflux activity were evaluated in S. cerevisiae AD∆ strain, which is deleted in 7 major efflux pumps. In addition, localisation and functional activity of S. cerevisiae protein Yol07Cp (putative orthologs of ABCG2) was also investigated. Furthermore, the significance of cholesterol in plasma membrane for localisation of ABCG2 was investigated using an ergosterol deficient strain S. cerevisiae AD∆H∆, which can grow auxotrophically in presence of cholesterol. Along with functional protein expression these approaches emphasized on stable localisation of ABCG2 in plasma membrane. Results show that ABCG2 constructs are expressed in S. cerevisiae AD∆ regardless of presence of affinity or reporter tags. All tested mutant ABCG2 constructs and chimera protein were also expressed. However, none of the protein constructs localised to the plasma membrane. In addition, they were non-functional in intact cells. Confocal microscopy images of GFP-tagged constructs of wildtype or each mutant ABCG2 showed that the overexpressed protein is retained and concentrated in discrete intracellular location. Further evaluation of membrane fractions, obtained from ABCG2 overexpressing cells by sucrose gradient centrifugation, probed with organelle specific antibodies; and ABCG2-GFP overexpressing intact cells probed with organelle specific dyes showed that ABCG2 is juxtaposed to nucleus and is closely associated with it. This discrete juxtanuclear structure visualised by transmission electron microscope resembled the ERACs which are induced by overexpression of membrane proteins in S. cerevisiae. Furthermore, the presence of cholesterol did not aid to target ABCG2 to plasma membrane in S. cerevisiae AD∆H∆. In this study I have demonstrated that ABCG2 overexpression can be successfully achieved in S. cerevisiae AD∆ or S. cerevisiae AD∆H∆ regardless of modifications tested. However, these protein modifications or the presence of cholesterol predicted to target ABCG2 to plasma membrane did not aid to correct the aberrant localisation of ABCG2 in S. cerevisiae. It appears that in addition to the presence of cholesterol one or more factors may be required for the correct targeting of ABCG2 in S. cerevisiae.
dc.format.mimetypeapplication/pdf
dc.language.isoen
dc.publisherUniversity of Otago
dc.rightsAll items in OUR Archive are provided for private study and research purposes and are protected by copyright with all rights reserved unless otherwise indicated.
dc.subjectABCG2
dc.subjectSaccharomyces cerevisiae
dc.subjectABC transporters
dc.subjectmembrane protein
dc.subjectHeterologous expression
dc.titleHeterologous expression of membrane proteins in Saccharomyces cerevisiae
dc.typeThesis
dc.date.updated2014-10-28T02:19:52Z
dc.language.rfc3066en
thesis.degree.disciplineDepartment of Oral Science
thesis.degree.nameDoctor of Philosophy
thesis.degree.grantorUniversity of Otago
thesis.degree.levelDoctoral
otago.openaccessOpen
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