Award details

Dynamic LOPIT: A new approach to organelle proteomics and its application to the endocytic pathway of clathrin-deficient vertebrate cells

Reference	BB/D001242/1
Principal Investigator / Supervisor	Dr Antony Jackson
Co-Investigators / Co-Supervisors	Professor Julian Griffin, Professor Kathryn Lilley
Institution	University of Cambridge
Department	Biochemistry
Funding type	Research
Value (£)	283,002
Status	Completed
Type	Research Grant
Start date	01/12/2005
End date	31/05/2009
Duration	42 months

Abstract

We have produced a vertebrate cell-line derived from the chicken B cell-line DT40 in which clathrin expression can be tightly regulated. Using this cell-line, we will apply a novel proteomic technique we call localisation of organelle proteins by isotope tagging (LOPIT) to catalogue many of the major protein components (intrinsic, peripheral and lumenal) of the organelles along the endocytic pathway of these cells. LOPIT will then be used to record how the steady-state levels of these organelle proteins change when clathrin is selectively depleted from the cells - hence our use of the term dynamic LOPIT. The project should dramatically increase our understanding of the role played by clathrin in the maintenance of the endocytic organelles and provide a wealth of practical experience in developing and extending the newly established LOPIT technology so as to allow its application to problems in cell biology in which understanding dynamic changes in intracellular protein distribution is fundamental.

Summary

Proteins on the surface membrane of animal cells are continuously taken inside the cell by a process called endocytosis.The proteins are mainly concentrated into structures called clathrin coated-pits that invaginate into the cell and pinch off to form clathrin-coated vesicles (CCVs). Once inside the cell, the CCVs fuse with other membrane-bound compartments and the internalised material is further sorted by subsequent transport between these intracellular compartments collectively called the endocytic pathway. Clathrin is believed to play multiple roles within this pathway, but the details are not clear. Here we propose a new approach to understanding the role of clathrin within this pathway by exploiting two recent inovations from our respective groups. Firslty, we will use a cell-line where we are able to control the expression of clathrin. Thus we can grow these cells under conditions where clathrin is either present or is absent from the cells. Secondly, we we will apply a new technique we call Localisation of Organelle Proteins by Isotope Tagging (LOPIT). This is a method that allows large numbers of proteins from the internal compartments to be simultaneously identified and their intracellular compartments identified. We aim to apply this technique to the above cells under clathrin-expressing and clathrin-depleted conditions. This will allow us to identify many of the proteins whose internal distribution is perturbed by clathrin. It will be the first time that the effect of removal of a single transport protein such as clathrin has been studied in such a comprehensive manner and should dramatically increase our understanding of how the endocytic pathway is maintained.

Committee	Closed Committee - Biochemistry & Cell Biology (BCB)
Research Topics	Technology and Methods Development
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

I accept the terms and conditions of use (opens in new window)

export PDF file

back to list new search