Protein Protocols Protein Protocols

Analysis of Isoprenylated Proteins 659
respective allylic pyrophosphate intermediates by two successive monophosphorylation
reactions (23). Further work is certainly warranted on the isolation and characterization
of these enzymes. The early developments in understanding the mechanism and
physiological significance of the salvage pathway for the utilization of F-OH and
GG-OH have been reviewed (24).
1.2. Experimental Strategy
Utilizing free F-OH or GG-OH as the isotopic precursors has several experimental
advantages over metabolic labeling of isoprenylated proteins with mevalonate. First,
F-OH and GG-OH are more hydrophobic and rapidly enter cultured mammalian cells.
They are efficiently utilized in a range of mammalian cell lines, and obviate the need to
include HMG-CoA reductase inhibitors to lower endogenous pools of mevalonate. The
experimental strategy is illustrated in Fig. 1. A key advantage of the strategy is that
F-OH and GG-OH are selectively incorporated into distinct subsets of isoprenylated
proteins, providing a simple and convenient approach to specifically label farnesylated
or geranylgeranylated proteins (Fig. 2).
Following metabolic labeling using [ 3H]F-OH, [ 3H]GG-OH, or [ 3H]mevalonolactone, the metabolically labeled proteins are exhaustively digested with Pronase E to liberate
the specific isoprenyl-cysteine residues (Fig. 3). The identity of the isoprenylated
cysteine residue can then be readily identified by normal or reverse-phase thin layer
chromatography (TLC). Figs. 4 and 5 show representative TLC analysis of isoprenyl-cysteines
released from metabolically labelled cellular proteins and from recombinant
proteins that were isoprenylated in vitro.
The isoprenol labeling and Pronase E methods may also be applied to the analysis of
individual metabolically labeled proteins. These methods provide a simple and convenient
approach for the identification of the isoprenyl group found on a specific protein. Two
experimental approaches are available. In the first, separate cell cultures are incubated
with [ 3H]mevalonate, [ 3H]F-OH, or [ 3H]GG-OH the metabolically labeled protein of
interest is then purified and subjected to sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) analysis. The specificity of the F-OH and GG-OH incorporation
allows the identity of the prenyl group to be directly assessed (Fig. 6). In the
second, the cells are metabolically labeled with [ 3H]mevalonolactone and the isolated
protein of interest is subjected to Pronase E treatment followed by TLC analysis of the
butanol-soluble products. In this case analysis of the chromatogram reveals the nature
of the isoprenyl group (Fig. 7). The experimental methods for these analytical procedures
are presented in detail in this chapter.
2. Materials
2.1. Metabolic Radiolabeling of Mammalian Cells in Culture
with [ 3H]Farnesol, [ 3H]Geranylgeraniol, and [ 3H]Mevalonolactone 1. ω,t,t-[1-3H]Farnesol (20 Ci/mmol, American Radiolabeled Chemicals, Inc., St. Louis, MO).
2. ω,t,t,t-[ 3H]Geranylgeraniol (60 Ci/mmol, American Radiolabeled Chemicals, Inc., St.
Louis, MO).
3. [ 3H]Mevalonolactone (60 Ci/mmol, American Radiolabeled Chemicals, Inc., St. Louis, MO).
4. Appropriate cell culture media, plastic ware, and cell incubator.