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Full Description
It is hard to think of a protein in eukaryotic cells that does not undergo some type of posttranslational modification. The covalent attachment of l- ids to proteins, protein lipidation, occurs for a few thousand proteins. Several functions for protein lipidation are known. Protein lipids may target proteins to specific cellular membranes, they may serve as molecular switches that allow cytosol-to-membrane transfer, they may direct protein-protein compl- ation, and they may stabilize protein structure. In cases such as the fatty a- lation of intracellular loops of transmembrane proteins, the funtions of the protein lipidations are not known. This volume-Protein Lipidation Protocols-provides detailed meth- ologies for the study of these processes. Since this is a rapidly growing field, many new experimental techniques have been developing over the past few years. All of the experimental techniques described in this volume have emerged during this time. The editor has made a special effort to include only those techniques that have not been previously described in a "hands-on" f- mat.
Contents
In Vitro Analysis of GPI Biosynthesis in Mammalian Cells.- Selection of Mammalian Cell Mutants in GPI Biosynthesis.- Analysis of the Cell-Surface Distribution of GPI-Anchored Proteins.- Imaging Fluorescence Resonance Energy Transfer as Probe of Membrane Organization and Molecular Associations of GPI-Anchored Proteins.- Purification of Caveolae-Derived Membrane Microdomains Containing Lipid-Anchored Signaling Molecules, Such as GPI-Anchored Proteins, H-Ras, Src-Family Tyrosine Kinases, eNOS, and G-Protein ?-, ?-, and ?-Subunits.- Analysis of Lipids in Caveolae.- Analysis of the Carbohydrate Components of Glycosylphosphatidylinositol Structures Using Fluorescent Labeling.- Analysis of the Lipid Moiety of GPI Anchor in the Yeast Saccharomyces cerevisiae.- Rapid Identification of Cysteine-Linked Isoprenyl Groups by Metabolic Labeling with [3H]Farnesol and [3H]Geranylgeraniol.- Incorporation of Radiolabeled Prenyl Alcohols and Their Analogs into Mammalian Cell Proteins.- Reconstitution of Yeast Farnesyltransferase from Individually Purified Subunits.- Probing the Role of H-Ras Lipidation for Signaling Functions in Xenopus laevis Oocytes.- Fluorescence Measurement of Lipid-Binding Affinity and Interbilayer Transfer of Bimane-Labeled Lipidated Peptides.- Determination of the Kinetics of Intervesicle Transfer and Transbilayer Diffusion of Bimane-Labeled Lipidated Peptides.- Preparation and Assay of Myristoyl-CoA:Protein N-Myristoyltransferase.- Metabolic Labeling of Protein-Derived Lipid Thioesters in Palmitoyl-Protein Thioesterase-Deficient Cells.- Fatty Acid Analysis of Protein-Derived Lipid Thioesters Isolated from Palmitoyl-Protein Thioesterase-Deficient Cells.
