Protein palmitoylation is a reversible post-translational lipid modification that allows regulated tethering of proteins to membranes and many of key proteins of cellular signaling, cancer, neuronal transmission, and membrane trafficking are palmitoylated. Despite its well-recognized importance, palmitoylation remains surprisingly understudied. Palmitoylation is difficult to detect and predict. Furthermore, until quite recently, the enzymology of palmitoyl addition had remained a total black box. The first two palmitoylation enzymes, i.e. protein acyl transferases (PATs), were identified only four years ago by work in yeast. The two identified yeast PATs together pointed towards the DHHC protein family as a possible PAT family. Yeast has seven of these DHHC proteins, while 23 are identifiable from the human genome. To facilitate global approaches to palmitoylation, we have developed a new proteomic methodology that purifies and identifies palmitoyl- proteins from complex protein extracts. This approach has first been used in yeast to identify many new palmitoyl-proteins and also to match the various palmitoyl-proteins with their cognate modifying PAT. A new project will apply the same proteomic technologies towards an understanding of palmitoylation in the mammalian brain. Palmitoylation plays a prominent role both in the regulation of synaptic signaling as well as in neuronal development and plasticity. Like the yeast analyses, these first mammalian analyses likely will highlight unanticipated new roles for palmitoylation in the brain, providing stimulus to future neuroscientific research. In addition, this technology also will be used to identify the neuronal palmitoylation substrates of HIP14, a DHHC PAT that has been implicated as participating in Huntington's disease. A final project will build on a recently discovered role for palmitoylation in the quality control mechanisms that act at the endoplasmic reticulum (ER). Of particular interest will be an investigation of the role of palmitoylation in the quality control mechanisms that underlie pathogenesis of demyelinating, neurodegenerative Pelizaeus- Merzbacher disease. Thus, in addition to expanding basic understanding of the cell biology of this lipid modification, we also hope to gain insight into the role of palmitoylation in neurodegenerative disease.