Protein prenylation is the posttranslational attachment of either a farnesyl group or a geranylgeranyl group via a thioether linkage (-C-S-C-) to a cysteine at or near the carboxyl terminus of the protein. Farnesyl and geranylgeranyl groups are polyisoprenes, unsaturated hydrocarbons with a multiple of five carbons; the chain is 15 carbons long in the farnesyl moiety and 20 carbons long in the geranylgeranyl moiety (see Table 1). There are three different protein prenyltransferases in humans: farnesyltransferase (FT) and geranylgeranyltransferase 1 (GGT1) share the same motif (the CaaX box) around the cysteine in their substrates, and are thus called CaaX prenyltransferases, whereas geranylgeranyltransferase 2 (GGT2, also called Rab geranylgeranyltransferase) recognizes a different motif and is thus called a non-CaaX prenyltransferase 1. Each protein consists of two subunits, α and β; the α subunit of FT and GGT1 is encoded by the same gene, FNTA (see Table 1).

The genomic organization of the human genes that encode protein prenyltransferases is shown in Figure 1. It is interesting to note that the genes encoding both subunits of non-CaaX prenyltransferases are much smaller (about 6-9 kilobases, kb) than those of the CaaX prenyltransferases (about 30-76 kb). The number of exons in the two types of genes are similar; the differences in gene size therefore result from differing intron lengths.

Automatic comparisons of data from expressed sequence tags (ESTs) with genes (for example using the program Acembly, for which the results are available from the NCBI AceView server 2) shows that all the human protein prenyltransferase genes have multiple alternative splice variants. The extent of translation of the various predicted transcripts and the structures and functions of the resulting proteins remain to be established experimentally; some of the predicted transcripts may be derived from missplicing rather than being real splice variants.

Protein prenyltransferases are currently known only in eukaryotes, but they are widespread, being found in vertebrates, insects, nematodes, plants, fungi and protozoa, including several parasites. The chromosomal locations and number of exons from protein prenyltransferase genes in the major eukaryotic model organisms are shown in Table 2. The α and β subunits have different folds, so are unlikely to have arisen from a common ancestor. Molecular phylogenetic analysis shows that orthologous proteins in different species are more closely related to each other than to their paralogs in the same species; the relationships between the β subunits are not fully clear. As well as the known genes, processed pseudogenes resulting from retrotransposition events have been found in the human and mouse genomes 34.

The α subunits of protein prenyltransferases consist of tetratricopeptide repeats and are part of the tetratricopeptide repeat superfamily 5, which also includes functionally diverse proteins involved in transcription, co-chaperoning, protein transport, cell-cycle control and phosphorylation. Although evolution of repeat proteins is difficult to analyze and interpret, Zhang and Grishin 6 have deduced convincingly that the FNTA and RABGGTA genes originated from a common ancestor that already contained multiple tetratricopeptide repeats rather than having independently amplified the number of motifs as the families diverged over time.

The evolutionary history of the β subunits of protein prenyltransferases has received much less attention so far than that of the α subunits 7. They can, however, be shown using standard sequence analysis tools such as PSI-Blast 8 or HMMer 9 to be part of a superfamily of prenyltransferases. Whereas the β subunits of protein prenyltransferases attach lipid anchors to proteins, other members of the superfamily catalyze different reactions involving polyisoprenes, such as the cyclization of polyisoprene derivatives during the synthesis of hopanoids in bacteria 10, cycloartenol in plants 11, ergosterol in fungi 12 and lanosterol in vertebrates 1314; these products are then generally processed further to produce essential substances such as cholesterol, steroid hormones or vitamin D and their equivalents. A merged PFAM 15 domain (PF00432) has been created containing the β subunits of protein prenyltransferases, as well as squalene-hopene cyclases and lanosterol and cycloartenol synthases. Intriguingly, structural superposition of the conserved (α-α)₆ barrel forming one half of bacterial squalene-hopene cyclase (the enzyme that synthesizes hopanoids) 16 with the β subunit of rat FT 17 reveals correspondence not only of their secondary structural elements but also of the parts of their active or binding sites (Figure 2). This would make sense if the different enzymes have retained common reaction mechanisms - or at least common substrate-binding characteristics - during evolution. Indeed, all of the members of the prenyltransferase superfamily bind substrates containing isoprenyl units, although they bind different numbers and variations of such units and the underlying processing and binding mechanisms seem to have diverged widely over time.

CaaX prenyltransferases recognize the carboxy-terminal Ca₁a₂X motif (see Table 1) 2324 of substrate proteins, usually after binding farnesyl-pyrophosphate 25 or geranylgeranylpyrophosphate 26. The lipid anchors are then transferred by a catalytic mechanism that depends on formation of a complex between a Zn²⁺ cation and the cysteine of the Ca₁a₂X motif 20. High concentrations of Mg²⁺ are required for optimal enzymatic activity of FT 27, though this is apparently not the case for GGT1 28. The Zn²⁺ is suggested to be required for the proper conformation of the substrate peptide 17. The major conformational change in the transfer step seems to be a rotation of the prenylpyrophosphate in the binding pocket and not of parts of the enzyme itself. A detailed picture of the reaction pathway that involves electrophilic and nucleophilic mechanisms is given by a series of structures representing the different states 28 as well as by kinetic measurements 29.

In contrast to FT and GGT1, GGT2 does not require a very specific carboxy-terminal motif 30 apart from the availability of several cysteines close to the carboxyl terminus that are often arranged -CC, -CXC, -CCX, -CCXX, -CCXXX or, in a few cases, with only a single cysteine as in -CXXX. If the motif consists of two cysteines in close proximity, two geranylgeranyl moieties are usually added.

GGT2 recognizes the structural features of a complex of the substrate with an escort protein (Rab escort protein (REP), previously known as component A) and then scans the carboxyl terminus for prenylatable cysteines 30. The catalytic mechanism of lipid transfer from geranylgeranylpyrophosphate to the protein substrate also requires Zn²⁺, and the following model has been presented for how the double geranylgeranylation could take place on the basis of insights from the reaction pathway of FT 28. After attachment of the first prenyl group, the lipid chain is translocated over the enzyme surface into another hydrophobic groove upon binding of the second geranylgeranylpyrophosphate. Finally, binding of a third geranylgeranylpyrophosphate releases the whole complex of the now doubly geranylgeranylated substrate with its escort protein; the escort protein is also involved in the transport of the substrate to the target membrane 3132.

The results of systematic oligonucleotide microarray experiments catalogued in the human gene-expression index database 3334 show that protein prenyltransferases are expressed in a variety of tissues. Both CaaX prenyltransferases seem to be active (that is, their α and β subunits are coexpressed) in a range of tissues, and the non-CaaX prenyltransferase subunits are also expressed in several more tissues. It should be noted, however, that many of the expression levels listed 3334 are close to the detection threshold and could therefore result from cross-hybridization between close homologs rather than true expression. Interestingly, the α subunits also appear to be expressed in tissues that lack expression of the corresponding β subunits; this suggests that single subunits, or isoforms of them, might have additional, prenylation-independent functions in the cell.

As the α subunit of CaaX prenyltransferases is shared between FT and GGT1, its expression must be higher than that of each β subunit if it is to form 1:1 complexes with the β subunits of both enzymes. These higher expression levels seem to be transcriptionally regulated by different promoters; in order to produce recombinant CaaX prenyltransferases in the laboratory, it is thus necessary to downregulate expression of the α subunits when coexpressing with β subunits 35.

Lipid anchors are common posttranslational modifications that can direct the subcellular localization of proteins. Other lipid modifications, such as myristoylation 363738, palmitoylation 3940 and glycosylphosphatidylinositol (GPI) anchors 4142, are mainly important for attachment of the protein to membranes, but lipid modification by protein prenyltransferases seems to have a more complex role: the farnesyl and geranylgeranyl moieties attached to the substrates are directly involved in protein-protein interactions as well as in protein-membrane interactions 43. The importance of protein prenyltransferases is illustrated by the involvement of their substrates in critical cellular pathways and diseases 44.

Typical substrates that are farnesylated by FT include many members of the Ras superfamily of small GTPases (H-Ras, K-Ras, N-Ras, Ras2, Rap2, RhoB (which is also geranylgeranylated), RhoE, Rheb, TC10, and TC21), as well as the nuclear lamina proteins lamin A and B, the kinetochore proteins CENP-E and CENP-F, fungal mating factors, cGMP phosphodiesterase α, γ subunit variants of G proteins, DnaJ heat-shock protein homologs, rhodopsin kinase, the peroxisomal membrane proteins Pex19 and PxF and paralemmin (a neural protein suggested to be involved in membrane dynamics). GGT1 preferentially geranylgeranylates some of the other small GTPases (such as Rac1, Rac2, RalA, Rap1A, Rap1B, RhoA, and RhoB (which is also farnesylated, as noted above), RhoC, Cdc42, Rab8 (which is also geranylgeranylated by GGT2), Rab11, and Rab13, as well as some γ-subunit variants of G proteins, cGMP phosphodiesterase β and the plant calmodulin CaM53. Typically, prenylation by CaaX protein prenyltransferases is accompanied by further posttranslational processing, most often involving cleavage of the carboxy-terminal tripeptide (-a₁a₂X) followed by carboxymethylation of the carboxyl terminus 454647. Palmitoylation is another modification that sometimes takes place after prenylation 48.

Because several prenylated substrates are involved in diseases, inhibition of protein prenyltransferases has great potential for medical applications. A boom in the field was triggered by the finding that inhibition of FT in mice that have tumors derived from H-Ras-transformed cells leads to tumor regression, while the inhibitor has no adverse effect on the organism 49. This led to successful completion of clinical phase I trials of farnesyl transferase inhibitors (FTIs), but in phase II trials the efficacy of the inhibitors towards a broad spectrum of different cancer cells (such as K-Ras-transformed cells) was far below the high expectations that arose from the phase I trials. Surprisingly, however, beneficial effects were found for other, non-neoplastic diseases; for example, diabetic retinopathy and macular degeneration 50. The unexpected physiological effects of FT inhibition are partly due to a striking cross-specificity between the two CaaX prenyltransferases: both FT and GGT1 can use either farnesyl-pyrophosphate or GGPP to a certain extent to transfer lipids to several of each others' substrates as well as their own 5152, and several substrates can be either farnesylated or geranylgeranylated. The substrates probably compete in vivo for the enzymes loaded with the preferred polyprenylpyrophosphate, and the type of modification that is added depends on the relative affinity of the substrates for the enzymes.

In cancer cells, FTIs are known to affect growth in soft agar (anchorage-independent growth), cell-cycle progression at the G1-S phase and the G2-M phase checkpoints, the morphology of the cytoskeleton (formation of actin stress fibers) and induction of apoptosis. The substrate proteins that have been suggested to be involved in these effects include H-Ras 49, Rheb 53, CENP-E 54 and RhoB 55; FTI effects are reviewed in more detail elsewhere 56. The effects of FT inhibition in cells transformed with H-Ras differ from those on cells transformed with K-Ras. This difference has been attributed to the ability of GGT1 to alternatively prenylate some proteins, through the cross-specificity mentioned above, including K-Ras but not H-Ras (see Figure 4). RhoB can be both farnesylated and geranylgeranylated; Prendergast and colleagues 57 have suggested that the different levels of farnesylated and geranylgeranylated RhoB in FTI-treated cells compared with untreated cells could contribute to the observed effects of FTIs.

The main substrates for prenylation by GGT2 are the Rab family of proteins, the largest group of small GTPases in the Ras superfamily. There are at least 60 different Rabs in humans 58. They interact with the Rab escort protein REP, which is required for the prenylation of Rabs by GGT2 30, and are involved in the docking of transport vesicles to their specific target membranes 59.

As with CaaX protein prenyltransferases, deficiencies in prenylation by non-CaaX protein prenyltransferases are relevant to diseases 5960. A mutation inactivating a start codon of the major transcript of the α subunit of GGT2 is one of the many mutations involved in the recessively inherited Hermansky-Pudlak syndrome and related disorders 6162, in which platelet synthesis, platelet organelle function and pigmentation are affected. X-linked choroideremia (CHM) results in retinal degeneration, with symptoms starting from night blindness in young people and progressing over decades until vision is completely lost 63. It is caused by loss-of-function mutations in the CHM gene, which encodes Rab escort protein 1 (REP1) 64. Loss-of function mutations in the Rab27a gene cause Griscelli syndrome, whose symptoms are similar to Hermansky-Pudlak syndrome and other diseases associated with insufficient Rab prenylation 60.