Identification of Novel Mitotically-Regulated Golgi Proteins

Lead Research Organisation: University of Manchester
Department Name: Life Sciences

Abstract

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.

Technical Summary

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.

Publications

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Title Antibodies to Golgi apparatus proteins 
Description Polyclonal antibodies to a variety of Golgi apparatus proteins. These are ZFPL1, GM130, GMAP-210. 
Type Of Material Antibody 
Year Produced 2007 
Provided To Others? Yes  
Impact It has helped us to publish several manuscripts and contributed to the research of other labs in the UK and abroad. 
 
Title Antibodies to Golgi proteins 
Description Antibodies to Golgi proteins TMF1, ZFPL1 and GMAP210 
Type Of Material Antibody 
Year Produced 2008 
Provided To Others? Yes  
Impact Not yet 
 
Title Cell lines stably expressing Golgi apparatus proteins 
Description Cell lines stably expressing GFP-tagged versions of the Golgi proteins ZFPL, GMAP210 and GM130. 
Type Of Material Cell line 
Year Produced 2008 
Provided To Others? Yes  
Impact None yet 
 
Title HeLa derived stable cell lines 
Description HeLa derived cell lines stably expressing versions of the novel Golgi protein ZFPL1. 
Type Of Material Cell line 
Provided To Others? No  
Impact Allowed us to publish EMBO J paper on functions of ZFPL1 
 
Title New solid phase protein binding assay 
Description It is a solid-phase binding assay for the analysis of protein-protein interactions. 
Type Of Material Technology assay or reagent 
Year Produced 2008 
Provided To Others? Yes  
Impact It allowed us to perform more quantitative binding assays, contributing to publications. 
 
Title Viral secretion marker for cell biology 
Description It is a viral vector for the expression of tsO45G into a multitude of mammalian cells. TsO45G is a commonly used secretory marker. Our vector allows efficient delivery into cells that are undergoing a wide variety of treatments. 
Type Of Material Technology assay or reagent 
Year Produced 2007 
Provided To Others? Yes  
Impact It helped us to publish 2 manuscripts on Golgi trafficking. 
 
Description Collaboration on GM130 and EspG during pathogenic E coli infection 
Organisation Imperial College London
Department Faculty of Natural Sciences
Country United Kingdom 
Sector Academic/University 
PI Contribution I contributed various reagents including constructs and antibodies and contributed to the experimental design.
Collaborator Contribution They performed the experiments and wrote the paper.
Impact Publication
Start Year 2010
 
Description GBF1 ARF collaboration 
Organisation University of Alabama at Birmingham
Country United States 
Sector Academic/University 
PI Contribution We identified the ARF proteins bound by the exchange factor GBF1. This was an important part of the published study.
Collaborator Contribution Scientific collaboration on existing projects
Impact Publication: PubMed ID 17956946
Start Year 2006
 
Description ZFPL1 and GMAP210 collaboration 
Organisation National Center for Scientific Research (Centre National de la Recherche Scientifique CNRS)
Department Laboratory of Molecular and Structural Virology CNRS
Country France 
Sector Academic/University 
PI Contribution We will provide recombinant proteins for use in assay, and perform assay under guidance of collaborator.
Collaborator Contribution To investigate membrane tethering in a specialised assay.
Impact None yet.
Start Year 2009