Reversible S-palmitoylation of cysteine residues critically controls transient membrane tethering of peripheral membrane proteins. Little is known about how the palmitoylation machinery governs their defined localization and function. We monitored the spatially resolved reaction dynamics and substrate specificity of the core mammalian palmitoylation machinery using semisynthetic substrates. Palmitoylation is detectable only on the Golgi, whereas depalmitoylation occurs everywhere in the cell. The reactions are not stereoselective and lack any primary consensus sequence, demonstrating that substrate specificity is not essential for de-/repalmitoylation. Both palmitate attachment and removal require seconds to accomplish. This reaction topography and rapid kinetics allows the continuous redirection of mislocalized proteins via the post-Golgi sorting apparatus. Unidirectional secretion ensures the maintenance of a proper steady-state protein distribution between the Golgi and the plasma membrane, which are continuous with endosomes. This generic spatially organizing system differs from conventional receptor-mediated targeting mechanisms and efficiently counteracts entropy-driven redistribution of palmitoylated peripheral membrane proteins over all membranes.

Cycles of depalmitoylation and repalmitoylation critically control the steady-state localization and function of various peripheral membrane proteins, such as Ras proto-oncogene products. Interference with acylation using small molecules is a strategy to modulate cellular localization--and thereby unregulated signaling--caused by palmitoylated Ras proteins. We present the knowledge-based development and characterization of a potent inhibitor of acyl protein thioesterase 1 (APT1), a bona fide depalmitoylating enzyme that is, so far, poorly characterized in cells. The inhibitor, palmostatin B, perturbs the cellular acylation cycle at the level of depalmitoylation and thereby causes a loss of the precise steady-state localization of palmitoylated Ras. As a consequence, palmostatin B induces partial phenotypic reversion in oncogenic HRasG12V-transformed fibroblasts. We identify APT1 as one of the thioesterases in the acylation cycle and show that this protein is a cellular target of the inhibitor.

A detailed study on the solid-phase synthesis of lipidated peptides of the Ras family employing the Ellman sulfonamide linker is reported. Using the C-terminal N-Ras sequence, critical issues such as lipidated amino acid resin loading, peptide elongation in the presence of labile groups and optimized conditions for release of the peptides were investigated. A versatile methodology for the synthesis of peptides with diverse lipid motifs and C-terminal methyl esters has accordingly been established.

Abstract In this review, an overview is given for the synthesis of lipidated peptides such as those belonging to the Rab, Ras, eNOS, and hedgehog proteins. The different approaches for the synthesis of specific lipidated peptides (palmitoylated, prenylated, and myristoylated) are discussed with special emphasis on solid‐phase synthesis methods, because these methods have turned out to be the preferred synthesis method for most required peptides. Different solid‐phase methods are described that are individually suited for different types of lipidated peptides, differing, for example, in lipidation pattern or amino acid side‐chain functionality and in their protein ligation functionality. For the described solution approaches, the block coupling strategies followed for the different lipidated peptides are highlighted. A special section in this review discusses the different synthetic tools for the incorporation of the lipid functionalities in the peptides. Although a generally validated strategy for the synthesis of lipidated peptides does not exist, because of the large number of different functionalities, this review aids in the conceptual design of synthetic protocols for lipidated peptides. Finally, the methods for the ligation of lipidated peptides to proteins are shortly discussed, as these methods also significantly influence the design and synthesis of lipidated peptides.

Biologically functional Ras isoforms undergo post‐translational modifications starting with farnesylation of the most C‐terminal cysteine. Combined with further processing steps, this isoprenylation allows for the anchoring of these proteins in endomembranes, where signal transduction events take place. The specific localization is subject to dynamic regulation and assumed to modulate the activity of Ras proteins by governing their spatiotemporal distribution. The δ subunit of phosphodiesterase (PDEδ) has attracted attention as a solubilization factor of isoprenylated Ras. In this study, we demonstrate that critical residues in the putative isoprenoid pocket of PDEδ can be mapped by coupling with a semisynthetic N‐Ras lipoprotein in which the native farnesyl group of the processed protein was replaced by a photoactivatable geranyl benzophenone moiety. The crosslinked product included parts of β‐sheet 9 of PDEδ, which contains the highly conserved amino acids V145 and L147. Modeling of the PDEδ–geranyl benzophenone (GerBP) complex supports the conclusion that the photolabeled sequence is embedded in the putative isoprenoid pocket of PDEδ.