Covalent attachment of fatty acids to proteins, a process known as acylation, is an important mechanism for targeting proteins to subcellular membranes or for enabling protein-protein interactions. In addition, acylation is critical for the correct functioning of many proteins. Acylation consists of two separate processes: myristoylation, the addition of a 14-carbon unsaturated fatty acid to a glycine residue at the amino terminus of the protein, and palmitoylation, the addition of a 16-carbon unsaturated fatty acid to a cysteine residue often near the amino terminus. This project will focus on myristoylation, which is catalyzed by N-myristoyltransferase (NMT). In animals and yeast, NMTs have been well characterized, and their protein sequences can be analyzed on-line to predict whether a particular protein will be a substrate for NMT. However, preliminary data using a direct assay for myristoylation showed that these databases often produced incorrect predictions for plant proteins. Therefore, plant NMTs probably have somewhat different substrate preferences than NMTs from yeast or animals. The long-term goal of this research is to understand the mechanism(s) of targeting myristoylated proteins to specific cellular locations and the impact of acylation on protein function. In order to accomplish this goal, it is important to accurately predict the plant proteins that are myristoylated. Preliminary data indicated that the Arabidopsis thaliana (mouse-eared cress) calcium-dependent protein kinase (CDPK) family would be a good set of test proteins because most of them are membrane-associated but do not contain transmembrane domains, an indication that acylation is the mechanism that they use for membrane binding. In addition, several CDPKs do not have predicted myristoylation sites (based on the yeast and animal models) although the CDPKs are myristoylated by direct assay. The two main objectives of this project are to determine the myristoylation status of selected CDPKs from Arabidopsis in order to clarify the substrate specificity of the plant NMT and to determine the importance of myristoylation for membrane binding. Myristoylation of CDPKs will be assessed in two ways. In the first method, in vitro myristoylation in a cell-free wheat germ extract will be measured. Second, for a subset of CDPKs, myristoylation will be confirmed by isolating affinity-tagged CDPK from plant extracts and subjecting the protein to mass spectrometry. In addition to detecting myristate groups, this method may also identify palmitate substitutions. To determine the importance of myristoylation in membrane binding, the glycine residue will be mutagenized at the site of myristate attachment, and the CDPK will be tagged with green fluorescent protein and used to generate transgenic Arabidopsis. Plants will be analyzed both by confocal laser scanning microscopy and by cell fractionation to determine how protein targeting is affected by blocking myristoylation. Undergraduate molecular biology students at the University of New Hampshire will be involved in preparing some of the site-directed mutants for use in this research. These studies will improve the ability to predict myristoylation of plant proteins and help to define the role of myristoylation in protein targeting to specific cellular membranes.
