Structural studies of glycoprotein sorting and processing complexes in the early secretory pathway

The secretory pathway of a eukaryotic cell exerts a stringently regulated quality control system for the correct folding and transport of new ly synthesised proteins and their subsequent transfer to their final destinations. Wh ile t is pathway has been mostly studied in yeast, it has become increasingly clear that aberra tions in its function are the main causes of a range of human disorders, and we are still in the p rocess of gathering knowledge of the underlying molecular structures and mechanisms of t he disease-causing agents in order to fully understand their impact. ERGIC-53, Erv41p and Ktr4p are all membrane anchored proteins involved in the transport or processing of lycoproteins, and form the main focus of this thesis. We have used X-ray crystallography and small-angle X-ray scattering, together with complementary biophysical and biochemical meth ods, to provide detailed descriptions of these proteins. The human glycoprotein transporter ERGIC-53 is resp on ible for the export of specific cargo proteins, which it binds in the endoplasmic reticul um (ER) and releases in the ER-Golgi intermediate compartment whilst cycling between the two organelles. Association of ERGIC-53 with the co-transporter MCFD2 is required for the transport of a subset of cargo proteins. ERGIC-53 is only able exert its function when present within the cell as a hexamer, but the details regulating its oligomeric state are still debated, and the structure of the hexameric protein remains unknown. We show that the oligomerisation of ERGIC-53 is independent of disulfide-bond formation and, based on small-angle X-ray scattering experiments, propose two alternative shapes describ ing the structure of the soluble lumenal part of the protein in its hexameric state. Erv41p is a glycoprotein transporter found in compl ex with its homologue Erv46p in yeast, and, similarly to ERGIC-53, the complex governs the export of cargo proteins from the ER to the Golgi apparatus, as well as the retrieval of es caped proteins back towards the ER. We have determined the structure of the soluble domain of Erv41p by X-ray crystallography, and show that the protein is comprised of a twisted β-sandwich. With almost the entirety of the concave face of Erv41p being negatively charged, th is could be the site of interaction with its cargo or another interaction partner. One cargo protein that has recently been revealed t o be ransported by the Erv41p/Erv46pcomplex is Ktr4p, a protein localised to the Golgi apparatus. Ktr4p is a member of a protein family associated with glycoprotein processing. The structures of the Ktr4p apo-protein and its complex with GDP were determined by X-ray cryst allography and show that the protein is comprised of a central β-sheet surrounded by α-helices, and that it belongs to the GT-A fold class of glycosyltransferases In addition, we have biochemically characterised the protein's function and show that it indeed possesses mannosyl transferase activity. LIST OF SCIENTIFIC PAPERS I. Biterova, E. I. , Svärd, M. , Possner, D. D. D. , & Guy, J. E. (2013). Purification, crystallization and preliminary Xray crystallographic analysis of the lumenal domain of the ER-vesicle protein Erv41p from Saccharomyces cerevisiae. Acta Crystallographica. Section F, Structural Biolo gy and Crystallization Communications , 69(5), 544–546. http://doi.org/10.1107/S1744309113008063 II. Biterova, E. I. , Svärd, M. , Possner, D. D. D. , & Gu y, J. E. (2013). The crystal structure of the lumenal domain of Erv41p, a protei n involved in transport between the endoplasmic reticulum and Golgi apparat us. Journal of Molecular Biology, 425(12), 2208–2218. http://doi.org/10.1016/j.jmb. 2013.03. 024 III. Possner, D. D. D. , Claesson, M. , & Guy, J. E. (2015). Structure of the glycosyltransferase Ktr4p from Saccharomyces cerevisiae . PLoS ONE, 10(8), e0136239. http://doi.org/10.1371/journal.pone. 01362 39 IV. Possner, D. D. D. , Abate, F. & Guy, J. E. (2016). Trichoplusia ni ferritin: a warning of a recurrent crystallisation contaminant from the High Five expression strain. Manuscript. V. Possner, D. D. D. , Wigren E. , Guy J. E. & Lindqv ist Y. (2016). Recombinant production of human ERGIC-53 in insect cells yields as embly-competent protein. Manuscript.