Many proteins are concentrated on the plasma membrane at specialized subcellular regions like synapses and caveolae by virtue of their lipid modifications. S-palmitoylation, a common form of lipid modification, is unique in that it is reversible and dynamic, suggesting a modulatory role in signal transduction similar to phosphorylation. Recent data indicate that such regulation of PSD-95 in the synapse is critical for synaptic organization and function; mutations in genes regulating protein lipidation are known to result in severe neurodegenerative disorders, emphasizing the importance of their regulation to human health and disease. Although characterization of the enzymes for S-palmitoylation (Palmitoyl thio-Acyl Transferases, S- PATs) has been controversial, recent data from yeast indicate that such enzymes exist. Similar studies in vertebrate systems have not been as fruitful. The work outlined in this proposal aims to meet this need by adapting a novel form of gene-trapping in cultured haploid cells for fully automated readout in a high-throughput microscopy format. This assay system will elucidate the entire protein S-palmitoylation pathway on a cell-by-cell basis over millions of cells imaged and individually recorded per well plate and multiplexed against further relevant fluorescent reporter readouts.
The specific aims described herein are 1) to construct and characterize a system that will report the palmitoylation potential of living cells as determined by the subcellular localization of a GFP fused to an S-palmitoylation substrate (GFP:SPS) and to use this cellular system as the target population for gene trap experiments designed to identify genes critical for S-palmitoylation 2) to scale the search for S-PATs to the level of a high-throughput screen by utilizing a novel machine-vision algorithm and a high-throughput cellular imaging system. Development of this system using S-palmitoylation as the proof-of-concept model pathway will provide a foundation that can be extended to screens for other genes that regulate the subcellular distribution and concentration of a reporter gene.
| Zhang, Jun; Planey, Sonia L; Ceballos, Carolina et al. (2008) Identification of CKAP4/p63 as a major substrate of the palmitoyl acyltransferase DHHC2, a putative tumor suppressor, using a novel proteomics method. Mol Cell Proteomics 7:1378-88 |
| Prigozhina, Natalie L; Zhong, Ling; Hunter, Edward A et al. (2007) Plasma membrane assays and three-compartment image cytometry for high content screening. Assay Drug Dev Technol 5:29-48 |
