Post-Golgi trafficking in the mammalian secretory pathway

Miranda, Kevin Charles (2004). Post-Golgi trafficking in the mammalian secretory pathway PhD Thesis, Institute for Molecular Bioscience, The University of Queensland.

       
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Author Miranda, Kevin Charles
Thesis Title Post-Golgi trafficking in the mammalian secretory pathway
School, Centre or Institute Institute for Molecular Bioscience
Institution The University of Queensland
Publication date 2004
Thesis type PhD Thesis
Supervisor Dr Rohan Teasdale
Dr Jennifer Stow
Dr Sean Grimmond
Total pages 101
Language eng
Subjects 06 Biological Sciences
Formatted abstract
A protein's intracellular trafficking route is determined by intrinsic targeting information encoded within its sequence. This thesis is primarily concerned with signal-based sorting and post-Golgi trafficking of proteins in epithelial cells. Four projects are presented which share this theme but differ in experimental approaches. E-cadherin is a cell surface glycoprotein transported to the basolateral domain of epithelial cells, where in complex with various members of the catenin family, it forms the core of the adherens junction and is responsible for cell-cell adhesion. E-cadherin is a very important protein in epithelial cells, however, the mechanisms that ensure correct polarized delivery of E-cadherin to the basolateral membrane are unknown. The sorting of transmembrane proteins in secretory pathways for their delivery to endosomes, lysosomes or to the plasma membrane relies on short peptide motifs within their cytoplasmic domains. The two most well-characterized sorting signals include critical tyrosine residues or dileucine motifs. This project initially set out to identify the sorting signal responsible for the basolateral trafficking of E-cadherin. Initially, the membrane proximal region of E-cadherin was shown to contain the information required for its basolateral targeting based on the expression of chimeric proteins consisting of varying segments of E-cadherin's cytoplasmic domain fused to a reporter molecule. A putative dileucine sorting signal (S1), within this region, was confirmed to be the basolateral sorting signal for cadherins generally, based on site specific mutagenesis of S1 in expressed chimeras. Generation of missorted, mutant E-cadherinΔSl1then enabled analysis of post-Golgi trafficking of other cadherin-bound catenins. The results show that E-cadherin and ß-catenin are sorted and trafficked together but that p120ctn does not join the complex until it reaches the basolateral membrane. These findings are important contributions to our understanding of polarized trafficking and of cadherin function and cell adhesion. E-cadherin is only the tenth protein identified to utilize a dileucine motif for basolateral sorting. The molecular details by which dileucine motifs function in targeting and vesicular trafficking are still not well understood. One approach to gather more information about dileucine sorting signals is to investigate their presence and function throughout the genome. Therefore a combined computational and biological pipeline was created to identify more proteins which utilize this trafficking mechanism based on a small subset of the RIKEN mouse transcriptome dataset. A test run proved successful with the identification of 16 novel proteins that could potentially utilize such sorting signals. The RIKEN transcriptome offers the opportunity for further classification and analysis of groups of proteins representing cargo or machinery. In the final chapter of this thesis, this dataset is mined to collate a set of proteins predicted to be secreted from the cell. In addition to this, highly relevant functional and expression information was gathered for these proteins giving valuable biological insight into a largely novel dataset. Taken together these studies at the interface of bioinformatics and cell biology have generated novel findings and produced important resources for studies on protein targeting in epithelial cells.
Keyword Golgi apparatus
Proteins -- Physiological transport

Document type: Thesis
Collection: UQ Theses (RHD) - UQ staff and students only
 
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Created: Wed, 21 Dec 2011, 12:11:31 EST by Erica Wei on behalf of The University of Queensland Library