Identification of Novel Mitotically-Regulated Golgi Proteins

All animal cells are made up of different compartments (organelles), each with a unique composition and a specific function. One of these compartments is called the Golgi complex, a collection of flattened membrane sacks called cisternae that are layered on top of each other to form stacks. The Golgi apparatus has two functions: it is responsible for modifying sugar chains present on proteins and lipids; and the packaging of these molecules into transport vehicles for delivery to the cell surface or other compartments in the cell. Proper modification and delivery of proteins is of fundamental importance to the cell and defects in these processes are responsible for many types of disease. It is therefore important that we understand how the Golgi complex operates at the molecular level. This project aims to identify novel proteins that play key roles in Golgi trafficking and in maintaining the organisation of this organelle. We have established an experimental system that will allow us to identify interesting candidate proteins, and when this has been achieved a number of complimentary approaches will be used to characterise the functions of these proteins in detail. This work is expected to enhance our understanding of Golgi structure and function.

The Golgi apparatus lies at the heart of the secretory pathway, receiving the entire output of proteins and lipids from the endoplasmic reticulum and packaging them into carriers for delivery to their final destinations. The mammalian Golgi apparatus is characterised by its unique structure, comprising a series of stacked cisternae connected laterally to form a compact ribbon in the perinuclear region of the cell. The molecular mechanisms responsible for generation and maintenance of Golgi structure are poorly understood. It is also not clear how the Golgi apparatus undergoes disassembly and reassembly during mitotic division.
Mitotic Golgi disassembly and inhibition of protein trafficking is triggered by phosphorylation of a subset of Golgi proteins. These are excellent candidates for having important roles in Golgi organisation and regulation of protein trafficking. The aim of this project to build upon my initial studies to identify the full complement of mitotic Golgi phosphoproteins (13 out of 21 remain unidentified). A wide range of modern cell biological techniques will then be used to analyse a subset of these proteins, studying their role in generation and maintenance of Golgi structure and protein trafficking. Protein overexpression, RNAi-mediated depletion, and antibody microinjection will be used to analyse the function of each protein in vivo. Effects upon Golgi structure will be studied at high resolution using electron microscopy in collaboration with Dr John Lucocq at the University of Dundee. Electron microscopy will also be used to localise the proteins at the ultrastructural level. The mitotic regulation of each protein will be studied to identify the mitotic phosphorylation site(s) (in collaboration with Professor Wolf Lehmann at the DKFZ, Heidelberg) and the timing of phosphorylation relative to changes in Golgi morphology and protein trafficking during mitosis. Biochemical techniques will be used to identify the mitotic kinase(s) responsible for phosphorylation and phosphorylation site mutants will be expressed in cells to analyse the role of phosphorylation in vivo. Finally, protein biochemistry and yeast two-hybrid screening will be used to identify interacting proteins to gain insights how the proteins function at the molecular level.
These studies will increase our understanding of how Golgi architecture is generated and its relationship to protein trafficking. We will also gain a much better understanding of how structure and transport are regulated during mitosis, and how the Golgi undergoes mitotic division.