Award details

Investigation of the peroxisomal protein import machinery by cross-linking

Reference	BB/D000904/1
Principal Investigator / Supervisor	Professor Alison Baker
Co-Investigators / Co-Supervisors	Professor Alison Ashcroft, Dr Katherine Johnston, Dr Jeffrey Keen
Institution	University of Leeds
Department	Ctr for Plant Sciences
Funding type	Research
Value (£)	268,072
Status	Completed
Type	Research Grant
Start date	01/11/2005
End date	31/10/2010
Duration	60 months

Abstract

Peroxisomes are organelles that play diverse but crucial biochemical roles in different species. These include primary metabolism, detoxification reactions and generation of signalling molecules. In plants peroxisomes are involved in mobilisation of storage reserves, the photorespiratory carbon cycle, catabolism of branched chain fatty acids, defence against oxidative stress and formation of jasmonic acid, nitric oxide and indole acetic acid, all important signalling molecules involved in plant defence responses and development. Peroxisome functions depend on import of proteins across the boundary membrane. Although many of the proteins involved in the import process have been identified and a network of protein-protein interactions mapped, a mechanistic understanding of how peroxisome protein import actually works is still lacking. This is because in vitro biochemical systems are very hard to work with. Uniquely we have developed both an in vitro import system and methods for generating a translocation intermediate- an imported protein arrested on its way into the peroxisome. We have now developed techniques to generate specific cross-links between this protein and peroxisomal membrane proteins which are strong candidates for components of the import machinery. Furthermore, this procedure introduces biotin into the crosslink partner as an aid to purification and identification. This proposal seeks to build on these developments to dissect the sequence of interactions between an imported protein and the peroxisome protein import machinery. Initially the feasibility of using peroxisomes isolated from Arabidopsis cell suspension cultures or Brassica napus cotyledons instead of sunflower cotyledons for the cross-linking will be investigated, as this will have advantages in terms of subsequent identification of cross-linked partners. However if yields are insufficient the well established sunflower system will be used. The hypothesis that the translocation intermediatemakes contacts with different subsets of the import machinery according to its location will be tested by manipulating conditions in ways that are known to differentially affect the binding and translocation of peroxisomal proteins. This will include manipulation of ATP levels, temperature and the use of other reagents that may block specific steps in the import process. Changing patterns of cross-links should allow the identification of sequential interactions with proteins of the import machinery. The hypothesis that the transported protein first assembles as part of a multi-protein complex (the 'pre-implex') on the surface of the peroxisome will be tested. The pre-implex has been proposed as a model for peroxisome protein import but no definitive experiments to support or refute this model have been presented. Two strategies will be employed to identify as many cross-link partners as possible. Immunoprecipitations will be carried out using 5 different antibodies against potential components of the import machinery that are available in the laboratory, plus any others that may become available during the lifetime of the project, either made in the laboratory or obtained from other laboratories. Cross-link partners that cannot be identified by immunoprecipitation will be affinity purified on avidin agarose using the introduced biotin tag. Initially peptide mass maps will be determined by MALDI-TOF MS. If it has proved possible to use Arabidopsis peroxisomes for the experiments the mass maps may be sufficient to identify the corresponding proteins, particularly as the size of the cross-linked protein may give important clues as to the likely identity. However, if no definitive matches are found (more likely if it is necessary to use sunflower peroxisomes) MS/MS sequence analysis will generate partial sequence to allow more refined data base searching and/or the design of oligonucleotides to isolate corresponding cDNAs by RT-PCR.

Summary

The aim of this project is to determine how proteins are transported (imported) into peroxisomes, which are a type of organelle (small structures) inside the cells of plants and animals. Understanding this process is crucial to understanding how peroxisomes function. Peroxisomes have several, vitally important biochemical roles. Genetic faults in peroxisomes cause developmental defects, including disability and death in humans. Peroxisome-deficient plants may not germinate or grow normally, or die as embryos. Peroxisomes need proteins to function, and all their proteins are imported from outside, across the membrane (a fatty barrier) that encloses the peroxisome. Protein transport across the membrane is carried out by a series of other proteins within it. About 30 such proteins are known, together with some of their interactions. For instance, receptor proteins (that bind to the proteins to be imported) have been identified. How they bind to the peroxisome membrane, and where they get the energy to do it, are known in outline. Some of these biochemical interactions have been worked out using purified peroxisomal proteins, but it is not feasible to study all the relevant proteins in this way. What is not available, and what we aim to provide, is an overall insight into the process of protein import, with the full sequence of protein interactions involved and the conditions required. My laboratory is the only one that has developed methods to track an imported protein (probe) as it moves through the membrane. We have also recently developed a way to chemically tag (label) the probe before it enters the peroxisome membrane and then transfer the tag to the proteins in the membrane with which the probe has interacted. The tagged proteins can then be sorted by gel electrophoresis, which separates molecules of different size, followed by western blotting, which reveals tagged proteins as a pattern of bands. By these methods, we will characterise the conditions underwhich the probe interacts with membrane proteins, by varying the amounts and types of biochemicals present. We will study the import of the probe in different experimental conditions that will alter the location of the probe in the membrane. For instance, we will record the changing pattern of labelling over time in the presence of the ATP (a molecule that provides the energy for protein import). As the probe moves through the membrane some contacts might be maintained, some lost and others gained, causing changes in the pattern of tagged bands on the Western blots. These experiments will clarify the changing environment of the probe as it moves from a surface bound location without ATP, to a membrane location with ATP. This will allow us to build up a picture of the steps involved in transport. We will use antibodies to distinguish any of the interacting proteins that are already known components of the import machinery from previously unknown ones. The unknown proteins will be isolated by affinity chromatography. They will then be identified after we have worked out their mass and their unique sequence of amino-acids. The importance of peroxisomes is outlined above. Their protein importation is less understood than almost any other membrane transport process, but it is known to be quite different from equivalent processes in other organelles. In other organelles, clarifying protein transport mechanisms has enabled major advances and applications. An equivalent understanding of peroxisomes could have similar benefits, for instance in treating certain human genetic disorders. In plants, it may be possible to manipulate or enhance the processes of seed dormancy, fat storage and germination, as well as the production of hormones, which influence how plants grow.

Committee	Closed Committee - Biochemistry & Cell Biology (BCB)
Research Topics	Plant Science
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

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