Christopher C. Farnsworth

Prenyl proteins in eukaryotic cells: a new type of membrane anchor

Recent studies have indicated that eukaryotic cells contain proteins that are post-translationally modified by long-chain, thioether-linked prenyl groups. These proteins include yeast mating factors, ras proteins and nuclear lamins. The modification occurs on a cysteine residue near the C terminus and appears to initiate a set of additional protein modification reactions that promote attachment of the proteins to specific membranes.

Disruption of Rab3-calmodulin interaction, but not other effector interactions, prevents Rab3 inhibition of exocytosis.

Rab GTPases regulate membrane traffic between the cellular compartments of eukaryotic cells. Rab3 is associated with secretory vesicles of neuronal and endocrine cells and controls the Ca2+‐triggered release of neurotransmitters and hormones. To clarify the mode of action of Rab3 we generated mutants of the GTPase that do not interact efficiently with its putative effectors Rabphilin and RIM. Surprisingly, these mutants transfected in PC12 cells were still capable of inhibiting Ca2+‐evoked secretion. Rab3 was shown previously to bind to calmodulin in a Ca2+‐dependent manner. By replacing two arginines conserved between Rab3 isoforms, we generated a mutant with a reduced affinity for calmodulin. This mutant retained the capacity to interact with the Rab3 regulatory proteins, Rabphilin, RIM, Mss4 and RabGDI, and was correctly targeted to dense‐core secretory granules. However, replacement of the two arginines abolished the ability of the GTP‐bound form of Rab3 to inhibit exocytosis of catecholamine‐ and insulin‐secreting cells. We propose that a Rab3–calmodulin complex generated by elevated Ca2+ concentrations mediated at least some of the effects of the GTPase and limited the number of exocytotic events that occurred in response to secretory stimuli.

Structural characterization of prenyl groups attached to proteins

Certain mammalian proteins are modified at a carboxyl-terminal cysteine by a thioether-linked prenyl group, the 15-carbon farnesyl or the 20-carbon geranylgeranyl moiety. Here, we describe analytical methods to determine the presence of a prenyl modification, the structure of the prenyl group, and the lipid: protein molar ratio for candidate proteins or their proteolytic fragments. Methods for the synthesis of prenyl standards are also presented. Methyl iodide or Raney nickel treatment is used to release the prenyl group from the protein for further analysis. When the prenyl group has been radiolabeled biosynthetically, two analytical techniques are available. Methyl iodide-released material, primarily the prenyl alcohol, can be cochromatographed with known standards using reverse-phase high-performance liquid chromatography to provide indirect structural data. Raney nickel-released material, primarily the unsubstituted hydrocarbon, can be analyzed by radiometric gas chromatography, which offers more precise structural information. However, unequivocal determination of the structure and quantitation of the mass of the released prenyl compound requires the use of gas chromatography-coupled mass spectrometry.

The prenylation of proteins

Proteins that are post-translationally modified by thioether-linked prenyl groups (long-chain isoprenoid products of mevalonic acid) include heterotrimeric and low-molecular-weight guanine nucleotide-binding proteins and nuclear lamins. In each case, a prenylated, C-terminal cysteine residue appears to influence protein location and activity in cells.

[16] Synthetic prenylated peptides: Studying prenyl protein-specific endoprotease and other aspects of protein prenylation

Publisher Summary This chapter describes the methodologies used for preparing synthetic prenylated peptides. These peptides are invaluable in studying the structures of prenylated proteins, the enzymes in the prenylated protein maturation pathway, and the binding of prenylated peptides to membranes. The methods discussed in the chapter are used to prenylate, site-selectively, any desired cysteine residue or residues in a peptide with multiple cysteines. The methylation of the C-terminal COOH group can be carried out without methylation of the side chains of aspartate and glutamate residues. Substituents can be introduced onto the N terminus of the peptides as well. The methods are also useful for preparing peptides that are radiolabeled to high specific activities. The use of such peptides to assay the prenylated protein-specific endoprotease (PPEP) is also described.

Biochemical adaptations of the retina and retinal pigment epithelium support a metabolic ecosystem in the vertebrate eye

Here we report multiple lines of evidence for a comprehensive model of energy metabolism in the vertebrate eye. Metabolic flux, locations of key enzymes, and our finding that glucose enters mouse and zebrafish retinas mostly through photoreceptors support a conceptually new model for retinal metabolism. In this model, glucose from the choroidal blood passes through the retinal pigment epithelium to the retina where photoreceptors convert it to lactate. Photoreceptors then export the lactate as fuel for the retinal pigment epithelium and for neighboring Müller glial cells. We used human retinal epithelial cells to show that lactate can suppress consumption of glucose by the retinal pigment epithelium. Suppression of glucose consumption in the retinal pigment epithelium can increase the amount of glucose that reaches the retina. This framework for understanding metabolic relationships in the vertebrate retina provides new insights into the underlying causes of retinal disease and age-related vision loss.

Exploring the specificity of prenyl protein-specific methyltransferase with synthetic prenylated rab peptides

Abstract Most Rab proteins contain two C-terminal geranylgeranyl groups. Some members are C-terminally m methylated, whereas others are not. Synthetic peptides bearing the sequence of the C-termini of Rab proteins and containing the C-terminal motif C(S-geranylgeranyl)AC(S-geranylgeranyl) are substrates for the membrane-bound prenyl protein-specific methyltransferase, whereas those that end in C(S-geranylgeranyl)C(S-geranylgeranyl) are not. Most Rab proteins contain two C-terminal geranylgeranyl groups. Some members are C-terminally methylated, whereas others are not. Synthetic peptides bearing the sequence of the C-termini of Rab proteins and containing the C-terminal motif C(S-geranylgeranyl)AC(S-geranylgeranyl) are substrates for the membrane-bound prenyl protein-specific methyltransferase, whereas those that end in C(S-geranylgeranyl)C(S-geranylgeranyl) are not.