Protein Prenylation: Genes, Enzymes, Targets, and Functions

Annual Reviews www.annualreviews.org/aronline

PROTEIN PRENYLATION: GENES, ENZYMES, TARGETS, AND FUNCTIONS

William R. Schafer and Jasper Rine Departmentof Molecular and Cell Biology, University of California, Berkeley, California 94720

KEYWORDS: Prenylation, posttranslational modification, isoprenoid, Ras proteins, palmitoylation

CONTENTS

INTRODUCTION ...... 209 GENES ENCODING PRENYLATED PROTEINS ...... 210 The Ras Superfamily ...... 210 Lipopeptide Pheromones ...... 213 Nuclear Lamins ...... 215 G-proteins ...... 216 PROTEINS INVOLVED IN PRENYL-DEPENDENTPROCESSING ...... 217 Protein Prenyltransferases ...... 218 Proteases ...... 221 Reversible Modifications ...... 223 PRENYL-MEDIATED INTERACTIONS ...... 225 Prenyl-protein Receptors ...... 225 Lipid Interactions ...... 227 Indirect Prenyl-dependent Targeting ...... 229

by Medical Research Council-Cambridge UK on 11/21/06. For personal use only. FUTURE PERSPECTIVES ...... 230 Annu. Rev. Genet. 1992.26:209-237. Downloaded from arjournals.annualreviews.org

INTRODUCTION

Theregulation, functional activation, andintracellular targeting of biological macromoleculesare often mediated through the covalent attachment of specific chemical moieties. The posttranslational modification of specific proteins by phosphorylation,glycosylation, acetylation, fatty acylation, and methylationhas been studied extensively, anda variety of critical biological functions have been ascribed to these particular side groups. In contrast to these familiar types of chemicalmodification, protein prenylationis a process 209 0066-4197/92/1215-0209502.00 Annual Reviews www.annualreviews.org/aronline

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which, until recently, was poorly understood. This article reviews the current understandingof the biological role for prenylation in the function of specific eukaryotic proteins, focusing on genetic studies of specific prenylatedproteins and the enzymesthat catalyze protein prenylation. In addition, we discuss possible models for howprenyl groups mediate specific protein functions, such as intracellular protein targeting. Wealso speculate on the possible relevance of protein prenylation to the medical problemsof oncogenesis and hypercholesterolemia. Prenylation refers to the covalent modification of a molecule by the attachment of a lipophilic isoprenoid group (reviewed in 26, 29, 46). Iso- prenoids are a diverse family of lipophilic molecules based on a repeating fwe-carbon structure. Famesyl diphosphate, a 15-carbon molecule, and geranylgeranyl diphosphate, a 20-carbon molecule, are the isoprenoid compounds most relevant to protein prenylation. Other biologically important isoprenoid molecules include cholesterol and other steroids, carotenoids, terpenes, and dolichols. A variety of small moleculesare prenylated, including quinones (coenzyme Q), porphyrins (heme a and chlorophyll), amino acids (dimethylallyl tryptophan), and purines (cytokinins). In addition, transfer RNAmolecules are prenylated at a specific adenine residue. All isoprenoids are synthesized from the six-carbon precursor mevalonatethrough a commonbiosynthetic pathway (47). Manyspecific proteins are posttranslationally modifiedwith prenyl groups. Most, if not all, prenylated proteins are modifiedby the attachment of either a 15-carbonfarnesyl or a 20-carbon geranylgeranyl group (117) in a thioether linkage to a cysteine residue. In most organisms, geranylgeranylation is a more commonmodification than farnesylation, although the relative propor- tions of farnesylcysteine and geranylgeranylcysteinein total cellular protein vary somewhatbetween organisms and cell types (37). It is possible that other types of prenyl modification of proteins mayexist; for example,a mammalian protein appears to be covalently modified with a prenylated adenine moiety (39). Prenylated proteins are found in a variety of cellular compartments,

by Medical Research Council-Cambridge UK on 11/21/06. For personal use only. including the nucleus, the cytosol, and membrane-boundorganelles (85, 129). Annu. Rev. Genet. 1992.26:209-237. Downloaded from arjournals.annualreviews.org

GENES ENCODINGPRENYLATED PROTEINS

Mostof the prenylated proteins whoseidentities have been determined belong to one of of four protein families: small GTP-bindingproteins, lipopeptide pheromones, nuclear lamins, and trimeric G-proteins. The importance of prenylation for the functions of these proteins is discussed below. The Ras Superfamily Most of the knownprenylated proteins are membersof the Ras superfamily of low-molecular-weightguanine nucleotide-binding proteins. These proteins Annual Reviews www.annualreviews.org/aronline

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can be divided into four smaller protein families: the Ras, Rho, Rap, and Rab/Yptfamilies (23). These proteins participate in a variety of cellular functions, including control of cell growth and differentiation, cytokinesis, and membranetrafficking. All small GTP-bindingproteins contain cysteine residues at or near the carboxyl-terminusthat appear to serve as targets for posttranslational prenylation. At least three distinct types of prenyl-dependent processing are involved in the maturation of these proteins, each of which affects a distinct subset of Ras-related proteins with a distinct carboxyl-terminal motif.

THECAAX MOTIF The Ras proteins are plasma membrane-localized molecules that regulate cell differentiation and proliferation in mammalianand yeast cells. Ras proteins contain a carboxyl-terrninal sequencecalled a CaaX motif, with a cysteine followed by two aliphatic residues and a carboxyl-terminal "X" residue, which can be C, S, M, Q, or A (135). Ras proteins undergo farnesylation of the cysteine residue (21, 54), proteolytic removalof the three amino acids distal to the cysteine, and methylation of the carboxyl-terminus (35, 50). SomeRas proteins are also palmitoylated at a cysteine residue near the famesylated cysteine (19). Ras proteins that lack this second cysteine residue are not palmitoylated but contain instead a region of basic aminoacids in the hypervariable domainnear the carboxyl-terminus (54). Ras is synthesized on soluble cytoplasmic ribosomes (50, 130), and is localized to the plasma membraneindependently of the secretory pathway. The localization and functional activity of Ras proteins depends on the presence of the farnesyl group. Genetic or pharmacological inhibition of protein prenylation blocks the membraneassociation and biological activity of both humanand yeast Ras proteins (54, 124). However,since RAS2protein from Saccharomycescerevisiae lacking the modifiable cysteine retains some functional activity when overexpressed in vivo (34), it appears that the function of prenylation is primarily to direct the polypeptide to the correct intracellular location. Proteolysis and methylationappear to play lesser roles

by Medical Research Council-Cambridge UK on 11/21/06. For personal use only. in mediating the association of Ras proteins with membranes.Whereas human Annu. Rev. Genet. 1992.26:209-237. Downloaded from arjournals.annualreviews.org Ras proteins lacking these modifications exhibit a reduced affinity for membranes(52), mutant K-ras alleles that prevent proteolysis and methylation do not block the membraneassociation or the transforming activity of the K-ras protein in vivo (71). Similarly, yeast mutations that cause a defect carboxymethylationdo not block Ras localization or functional activity (63, 90). In addition to the farnesyl group, either a palmitoylationsite or a polybasic region is required for efficient localization to the plasmamembrane and is important for the transforming activity of oncogenicmutant proteins (55).

THECAAL MOTIF A second group of small GTP-binding proteins contain a carboxyl-terminal motif similar to a CaaXbox, called a CaaLmotif, with a Annual Reviews www.annualreviews.org/aronline

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leucine residue in the carboxyl-terminal position. This group includes many proteins implicated in cytokinesis, such as the Rapand Rho protein families. Manyof these proteins are knownto be geranylgeranylated, including G25K (143), RhoA(70), Ral, Rac (74), Rapla (18), and Raplb (72). these proteins are also knownto undergo carboxyl-terminal proteolysis and carboxymethylation (18, 70, 72). Geranylgeranylation of these proteins widely thought to mediate association with intracellular membranes;however, only in the cases of Rapl and Rho has this hypothesis been supported experimentally. Rapla and Raplb are a pair of closely related genes that are membersof the Ras family of small GTP-bindingproteins (108). Overexpressionof Rap 1 (also knownas K-rev-1) results in suppression of the transforming activity activated Ras alleles (76). Unlike Ras proteins, whichare localized to the plasma membrane,Rap 1 proteins are associated with the Golgi complex(13). The posttranslational processing at the carboxyl-terminusof the Rapl b protein has been investigated in detail. The carboxyl-terminus, whichhas the sequence K-A-R-K-K-S-S-C-Q-L-L,undergoes four modifications: (a) geranylgeranylation of the cysteine residue; (b) proteolytic removalof the three terminal amino acids; (c) methylation of the new carboxyl-terminus, and (d) phosphorylation of the distal serine residue by A-kinase (72). A prenylated carboxyl- terminus is required for the association of Raplb with membranes.Prenylation and phosphorylation are both required for interaction of Raplb with a factor knownas GDS,which stimulates GDP/GTPexchange (56), since prenylated, phosphorylated peptides corresponding to the Raplb carboxyl-terminus can competewith intact Raplb for interaction with GDS,whereas other peptides lacking either modification can not (131). The Rho proteins are also localized to the Golgi (91), and also appear require geranylgeranylation for association with membranesand with exchange factors. Mature RhoAprotein binds to membranes, and associates with two GDP/GTPexchange factors, the stimulatory factor GDSand the inhibitory factor GDI.In contrast, bacterially expressed RhoAprotein, which

by Medical Research Council-Cambridge UK on 11/21/06. For personal use only. lacks carboxyl-terminalprocessing, is defective in all three of these interac- Annu. Rev. Genet. 1992.26:209-237. Downloaded from arjournals.annualreviews.org tions (59). Prenylation maybe generally involved in mediating interactions betweenother small GTP-bindingproteins and exchange factors (7).

OTHERMOT1FS A third group of small GTP-binding proteins contain the carboxyl-termina] sequences C-C, C-X-C,or C-C-X-X.These proteins include most of the Rab/Yptproteins, whichare involved in regulating intracellular trafficking, and are present both in the cytoplasmand associated with distinct membranecompartments (9). Several of these proteins, including mammalian Rablb and Rab3a and YPT1, YPT3, and YPT5 of Schizosaccharomyces pombe,have been shownto be geranylgeranylated in vivo (73, 102); genetic Annual Reviews www.annualreviews.org/aronline

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and biochemical evidence indicates that others, including the mammalian Rablb, Rab2, and Rab5 (75), and YPTI of S. cerevisiae (119), are geranylgeranylatedas well. Using a variety of approaches, including mutation of the terminal cysteine residues (97), depletion of intracellular isoprenoid pools (73, 75), expression in bacterial cells (7), and analysis of geranylgeranyltransferase mutants (119), geranylgeranylation has been shownto essential for association of these proteins with membranes. Although proteins containing C-C, C-X-C, and C-C-X-Xmotifs are all geranylgeranylated, someevidence indicates that the modifications directed by these sequences may differ in important respects. MammalianRab3A, which contains the C-X-Cmotif, is geranylgeranylated at both carboxyl-terminal cysteine residues and is carboxymethylated (38). This pattern modification maybe commonto all C-X-Cproteins; another protein containing this sequence motif, the YPT5protein of S. pombe, is also geranylgeranylated and carboxymethylated(102). In contrast, proteins with carboxyl- terminal C-C motifs maybe modified differently. The YPTIprotein of S. cerevisiae, with a C-C motif, is palmitoylated, and this palmitoylation is dependenton the presence of the carboxyl-terminal cysteines (97). However, no methylation of the C-C proteins YPT1and YPT3of S. pombeis detected under the labeling conditions used to detect methylationof Y PT5(102). Thus, C-C proteins maybe geranylgeranylated and palmitoylated, but not methylated. The sites of these modifications are undetermined.Since, in vitro, the yeast and humanmethyltransferases appear to methylate essentially any prenylcysteine with a free carboxyl (63, 132), the apparent lack of methylated carboxyl-terminussuggests that the carboxyl-terminal cysteine of C-C proteins maynot be prenylated. Thus, a reasonable hypothesis compatible with these data is that the carboxyl-terminal cysteine is palmitoylated, and that the adjacent cysteine is geranylgeranylated. A novel carboxyl-terminal sequence, C-C-P, is found in Rah, a mammaliansmall GTP-bindingprotein related to Rhoproteins (99). It is temptingto speculate that this mayrepresent a new prenylation motif. by Medical Research Council-Cambridge UK on 11/21/06. For personal use only. Annu. Rev. Genet. 1992.26:209-237. Downloaded from arjournals.annualreviews.org Lipopeptide Pheromones Haploid fungal cells secrete mating pheromonesthat trigger cell fusion with cells of the opposite matingtype to form diploid zygotes. Several fungal mating pheromonesare lipopeptides, consisting of an oligopeptide to whicha prenyl group is covalently attached in a thioether linkage to a carboxyl-terminal cysteine residue. The most extensively characterized is the mating pheromone a-factor of yeast. Yeast contain two a-factor genes, MFA1and MFA2,which encode polypeptide precursors of 36 and 38 amino acids, respectively, each with a carboxyl-terminal CaaXsequence (15, 93). The a-factor precursor undergoesextensive processing independentlyof the classic secretory pathway Annual Reviews www.annualreviews.org/aronline

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before being secreted as the mature 12-aminoacid lipopeptide (134). Analysis of the structure of maturea-factor reveals that four modificationsoccur in the processing of the precursor to yield active a-factor: (a) proteolytic removal of a 21-aminoacid pro sequence to yield the mature amino terminus of the peptide, (b) attachment of the famesyl group to a cysteine residue three amino acids from the carboxyl-terminus, (c) proteolytic removalof the three amino acids distal to that cysteine, and (d) methylation of the newly generated carboxyl-terminus (5). The four steps in a-factor maturation occur in a defined order. In vitro studies of a-factor precursor processing have demonstratedthat farnesylation is a precondition for carboxyl-terminal proteolysis (8) and methylation (61). Furthermore, cells depleted for mevalonate in vivo accumulate an a-factor species with the electrophoretic mobility characteristic of the a-factor primary precursor (124, 133), suggesting that farnesylation is a precondition for amino-terminalproteolysis. Thus, farnesylation is the obligatory first step in a-factor precursor processing, followed by proteolysis and methylation at the carboxyl-terminus, and proteolysis at the amino terminus. It is not known whether amino-terminal proteolysis and carboxyl-terminal proteolysis occur in a fixed order. Farnesylation and/or proteolysis is also apparently required for the secretion of a-factor, since unfarnesylated, unproteolyzed a-factor precursors accumulatein the cytosol (124), whereasunmethylated a-factor readily secreted (90). The farnesyl group is also important for the ability a-factor to induce growth arrest and morphologicalalteration of yeast cells of the a mating type (5). Interesting variations on this process are seen in the processing of other pheromones. Rhodotorucine A, a lipopeptide pheromonefrom the basidio- mycetousjelly fungus Rhodosporidiumtoruloides, is an 11-aminoacid peptide with a carboxyl-terminal cysteine covalently bound to a farnesyl group in a thioether linkage (69). In contrast to a-factor, the rhodotorucineA carboxyl- terminal cysteine is not methylated. Three genes, RHA1,RHA2 and RHA3, encode rhodotorucine A precursors consisting of three to five tandemcopies

by Medical Research Council-Cambridge UK on 11/21/06. For personal use only. of the rhodotorucine A peptide (2). These peptide sequences are separated Annu. Rev. Genet. 1992.26:209-237. Downloaded from arjournals.annualreviews.org a four-amino acid spacer peptide, (C)-T-V-A/S-K. The rhodotorucine precursor is apparently cleaved to produce multiple pheromonepeptides, a process reminiscent of a-factor processing in S. cerevisiae and neuropeptide processing in mammals.Maturation of rhodotorucine A may involve cleavage of the large precursor to producemultiple peptides containing canonical CaaX sequences, whichare then processedin a mannersimilar to a-factor precursor. Alternatively, farnesylation of internal cysteine residues could occur prior to proteolytic maturation. In either case, rhodotorucine A provides a precedent for prenylation of internal cysteine residues (see also Proteases below) Annual Reviews www.annualreviews.org/aronline

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The processing of the tremerogen A-10 pheromone,produced by the jelly fungus Tremella mesenterica, is another variation on the maturation process of a-factor. TremerogenA-10 is synthesized as a large precursor of unknown primary sequence. The secreted pheromone is a farnesylated, carboxymethylated, 10-aminoacid lipopeptide with an apparent molecular weight of 1.5 kd. Interestingly, the farnesyl groupcontains an alcohol group at position 30 (122). Compactin,an inhibitor of isoprenoid biosynthesis, inhibits maturation of tremerogenA-10 precursor, resulting in the accumulationof a 28-kd species that is presumablyunfarnesylated. However,tunicamycin, an inhibitor of glycosylation, and monensin,an inhibitor of Golgi transit, also inhibit processing, and result in the accumulation of lower molecular weight intermediates, implying that the precursor is glycosylated and processed in the Golgi (96). TremerogenA-10 is therefore a novel case of a farnesylated polypeptide that, unlike a-factor and the small GTP-bindingproteins, is also processed in the secretory pathway.Since farnesylation is an early processing step that may occur in the cytoplasm, prenylation may mediate entry of tremerogen A-10 into the endoplasmic reticulum. In addition to these examples, a wide variety of evolutionarily divergent fungal species secrete prenylated pheromones.For example, h" cells of the fission yeast S. pombesecrete a nine-amino acid methylated lipopeptide knownas M-factor, which is encoded by two genes, MFmland MFm2.The precursors encoded by these genes contain CaaXsequences, and appear to undergo the sameposttranslational modifications as a-factor (32). A number of mating factors from jelly fungi are prenylated peptides; some, such as tremerogen a-13 from T. mesenterica, contain a thioether-linked farnesyl group, whereas others, such as A-9291-I from T. brasiliensis, contain an oxidized farnesyl group (67, 69, 122). In addition, the mating type of the basidiomycetoussmut fungus Ustilago maydis is determined in part by which of two genes, mfal or mfa2, is present at the A mating-type locus; both mfal and tufa2 encode short polypeptides that contain canonical CaaXsequences (14). Thus, prenylated pheromonesmay be ubiquitous amongthe fungi. It

by Medical Research Council-Cambridge UK on 11/21/06. For personal use only. unclear whether prenylated peptide hormonesare found in other organisms. Annu. Rev. Genet. 1992.26:209-237. Downloaded from arjournals.annualreviews.org

Nuclear Lamins Laminsare nuclear proteins that are a major componentof the karyoskeleton. Lamins are thought to form intermediate filaments that are associated with the inner surface of the nuclear membraneand mediate nuclear breakdown and reformation during mitosis. Laminproteins fall into three types, A-type, B-type, and C-type. B-type lamins are always associated with the nuclear membrane,whereas A- and C-type lamins are found in a soluble form during mitosis (42). Both A- and B-type lamins undergoposttranslational processing Annual Reviews www.annualreviews.org/aronline

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involving prenylation, and both contain a carboxyl-terminal CaaXsequence, In B-type lamins, posttranslational processing resembles the processing of Ras proteins. The cysteine residue of the CaaXsequence is farnesylated (139), and the three aminoacids distal to that cysteine are removed(137). There also evidence that lamin B is carboxymethylated, although the methylation appears to be transient, and actually may play a role in regulating the disassembly of the nucleus during mitosis (25). As in the cases of Ras and a-factor, prenylation appears to be the first step in processingof lamin B, and a precondition for other processing steps (11,137). Since the presence of the CaaX sequence is necessary for nuclear membranetargeting of lamin B molecules(58), prenylation and perhaps proteolysis and methylation are also necessary for the targeting of the lamin proteins to the nuclear membrane. Depletionof nonsterol isoprenoid pools in animal cells causes cells to arrest specifically at the G1/Stransition in the cell cycle (111). it has been proposed (46) that this requirementcould in fact reflect a requirementfor lamins at this stage of the cell cycle. The processing of lamin A is more complex. The lamin A precursor is initially modified with a famesyl group to produce an intermediate knownas prelarnin A. Subsequently, however, the 18 carboxyl-terminal amino acids, including the farnesylated CaaXsequence, are removedby an endoprotease; thus, the mature form of lamin A that is assembled into the nuclear lamina is not prenylated (I 1). Mevalonate-starvedcells accumulate unfarnesylated lamin A precursor in nuclear inclusions, and this precursor is rapidly processed and assembled into the lamina upon addition of mevalonate. On the basis of this observation, it has been suggested that farnesylation of lamin precursors occurs not in the cytosol, but in the nucleus (82). Interestingly, the carboxyl-terminal region of the lamin A precursor is apparently not required for assemblyof lamin A into the lamina. This has led to the speculation that the prenylated lipopeptide released in lamin A processing mayitself have a biological function (82).

by Medical Research Council-Cambridge UK on 11/21/06. For personal use only. G-proteins Annu. Rev. Genet. 1992.26:209-237. Downloaded from arjournals.annualreviews.org G-proteins are heterotrimeric GTP-binding proteins that mediate signal transduction between transmembranereceptors and intracellular second mes- sengers. The ~/ subunits of trimeric G-proteins contain either CaaXor CaaL consensus sequences. Several of these ~/subunits are knownto be prenylated. The ~ subunit of bovine transducin, the G-protein involved in visual signal transduction, contains a farnesyl group covalently linked to the carboxyl-terminal cysteine residue (44, 80). Transducin ~/is also modified by proteolysis and carboxymethylation. In addition, STE18,the ~/subunit of the G-protein involved in yeast mating, requires a prenyl modification, probably farnesyl, for membranelocalization and functional activity (41). A numberof uniden- Annual Reviews www.annualreviews.org/aronline

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tified G~/ subunits from bovine brain and from rat tissue culture cells are geranylgeranylated (84, 100, 142). In the G~/ subunits of bovine brain, the prenylated cysteine is apparently carboxymethylated(142). The famesylation of bovine transducin is of particular interest because several other proteins involved in visual signal transduction in the rod outer segment (ROS) membraneare prenylated. For example, the cGMPphos- phodiesterase, which responds to activated transducin by degrading the second-messengercGMP, is a heterotetramer composedof an or, 13, and two ~/ subunits. The ot subunit of this enzymeis farnesylated and carboxymethylated, whereas the [3 subunit is geranylgeranylated, but apparently not methylated (4). In addition, rhodopsin kinase, which is involved in the attenuation of rhodopsin activation, is also famesylated and carboxymethylated (66). Rhodopsin itself contains a novel type of prenyl modification: the covalent attachment of 11-cis-retinal, a molecule derived from carotenoids, to a lysine residue of the opsin protein. Rhodopsinis also palmitoylated, a modification commonto manyprenylated proteins (104). Theinvolvement of several prenylated proteins in a. single signal transduction pathway suggests that this modification maymediate colocalization of and interaction betweenthese proteins. At least two Got subunits contain carboxyl-terminal sequencesthat resemble the CaaXmotif. However,neither of these proteins is prenylated in vivo (84, 123). Since mutant versions of these tx subunits containing the sequence C-V-L-Sin place of the normal sequence C-G-L-Fare prenylated (68), it has been suggestedthat the glycine residue prevents prenylation of these proteins. However, Ras mutant proteins with the sequence C-G-L-F are capable of undergoing prenylation in vivo (71). Therefore, it remains unclear whythis sequence does not direct prenylation of the Got subunit.

PROTEINS INVOLVED IN PRENYL-DEPENDENT PROCESSING by Medical Research Council-Cambridge UK on 11/21/06. For personal use only. Annu. Rev. Genet. 1992.26:209-237. Downloaded from arjournals.annualreviews.org The maturation of prenylated proteins involves manyvariations on a single theme, rather than a single series of processing reactions. All known prenylated proteins are initially modified by the attachment of a farnesyl or geranylgeranyl group in a thioether linkage to a cysteine residue at or near the carboxyl-terminus. Subsequently, manyof these proteins are further modifiedby proteolytic processing. Finally, the carboxyl-terminal regions of manyprenylated proteins are targets for reversible modifications such as palmitoylation, methylation, and phosphorylation. The specific proteins that direct these modifications are critical for mediating the commonand divergent biological properties of the processed carboxyl-termini. Annual Reviews www.annualreviews.org/aronline

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Protein Prenyltransferases

BIOCHEMICALSTUDIES Several enzymes, termed protein prenyltransferases, which catalyze the attachment of a specific prenyl moiety to specific protein precursors, have been identified and characterized. At least three distinct protein prenyltransferase activities are present in yeast and mammaliancells. Protein farnesyltransferases (PFT) are soluble enzymesthat catalyze the farnesylation of humanK-ras (86, 112), a-factor precursor of yeast (125), and RAS2of yeast (48). The substrates for these enzymesare the farnesyl donor farnesyl diphosphate (FPP) and a protein precursor containing an intact CaaXsequence. The farnesyltransferase in rat brain has been purified to homogeneity. The enzymeis a heterodimer composedof nonidentical o~ and 13 subunits; the genes encoding these proteins have recently been sequenced (113). A second activity, termed protein geranylgeranyltransferase I (PGT has also been identified in soluble extracts. This enzyme,which is chromatographically (22, 144) and genetically (40) separable from PFT, catalyzes geranylgeranylation of peptides containing CaaL sequences, using geranylgeranyl diphosphate (GGPP)as a substrate. This enzymehas been only partially purified. However,since antisera directed against the mammalian PFTot subunit can immunodepletePGT I activity from crude extracts, it has been hypothesized that mammalianPGT I is also a heterodimer with an ct subunit that is shared with PFT(127). A third enzyme,PGT II, catalyzes the geranylgeranylation of protein precursors with C-C carboxyl-terminal motifs (98). This enzymeis chromatographically separable from both PFT and PGTI. Activities have also been identified in mammaliancells that catalyze the geranylgeranylation of C-X-Cand C-C-X-Xmotifs (60, 75). Although both activities are separable from PFTand PGTI, it is not clear whether either of these activities is distinct from PGTII. In addition, the observation that farnesylation of prelamin A apparently occurs in the nucleus, whereas Ras farnesylation occurs in the cytoplasm, maysuggest the existence of a distinct nuclear farnesyltransferase (82). In this regard, it has been by Medical Research Council-Cambridge UK on 11/21/06. For personal use only.

Annu. Rev. Genet. 1992.26:209-237. Downloaded from arjournals.annualreviews.org reported that farnesyltransferase activity from bovineextracts fractionates as two peaks on gel filtration chromatography; perhaps one of these peaks correspondsto the putative lamin farnesyltransferase (86). The substrate specificities of the protein prenyltransferase enzymesare clearly critical for the attachmentof the correct lipid modificationto specific prenylated proteins. Current evidence suggests that the specificities of PFT and PGTI are largely determined by the particular amino acids present in the CaaX/CaaL motif itself. Comparison of the sequences of known farnesylated and geranylgeranylated proteins led to the hypothesis that the carboxyl-terminal amino acid of the protein precursor determined whether a particular protein was farnesylated or geranylgeranylated. According to Annual Reviews www.annualreviews.org/aronline

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this hypothesis, proteins containing S, C, M, Q, or A in this position are substrates for PFT, whereas proteins with L or F are substrates for PGTI. This hypothesis has been supported most convincingly for PFTin competition experiments using synthetic CaaX-related peptides (114). The in vitro specificity of PGTI also conforms largely to in vivo predictions (98). Interestingly, both PFr and PGTI can act weakly in vitro on the preferred substrates of the other enzyme(144). The PGTII enzymeshows stalking substrate specificity. Intriguingly, the sequences required for geranylgeranylation by PGTII, unlike PFTand PGT I, are not exclusively located at the carboxyl-terminus(98). Geranylgeranyla- tion by PGTII is not competedby peptides corresponding to the carboxyl- termini of PGTII substrates, nor are these carboxyl-terminal peptides used as substrates by the enzyme.Furthermore, protein precursors containing C-C carboxyl-termini are not weakly prenylated by PFF or PGTI. Thus, the recognition of protein substrates by PGTII maybe mechanistically dissimilar to substrate recognition by PFTand PGTI. The internal sequences required to direct geranylgeranylation by PGTII have not been identified.

GENETICSTUDIES In S. cerevisiae, studies of the genes encoding protein prenyltransferase subunits have been instrumental in revealing manyof the roles of protein prenylation. The yeast genes RAM1(125) and RAM2(48) required for PFTactivity. Bacterially expressed RAM1and RAM2proteins, upon mixing, can reconstitute active yeast PFTenzyme; thus, these proteins apparently comprise the subunits of the yeast heterodimer (57). The protein sequences of RAM1and RAM2are homologous to the mammalian PFT 13 and ot subunits, respectively (77). Mutations in RAM1have been isolated independently by several groups based on several phenotypes: suppression of RAS2activation (43, 109), a defect in the production of mature a-factor (138), and interference with the function of the G’~ subunit STE18(94). Thus, the yeast PFTis required for the functions of all the knownyeast farnesylated proteins (it is worthnoting that lamins have not been identified in yeast cells).

by Medical Research Council-Cambridge UK on 11/21/06. For personal use only. Mutations in RAM2also suppress RAS2activation and block production of Annu. Rev. Genet. 1992.26:209-237. Downloaded from arjournals.annualreviews.org active a-factor (57). Yeast contains two additional genes that share sequence homologywith RAM1.One of these is knownas CDC43or CALl. Mutations in this gene were identified on the basis of two distinct phenotypes, call mutants arrest specifically at the G2/Mtransition unless grownin the presence of high levels of calcium (106). These mutants are suppressed by overexpression of yeast (RHO2),which contains the carboxyl-terminal sequence C-I-V-L, suggesting RHOproteins as possible targets of the CDC43enzyme (Y. Ohya, personal communication). The cdc43 mutants, in contrast, display a temperature-sensitive defect in eytokinesis and the establishment of cell Annual Reviews www.annualreviews.org/aronline

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polarity (1). Mutations in CDC42and RSR1, which encode proteins that contain the carboxyl-terminal sequence C-T-I-L, also cause this phenotype, suggesting that these proteins are also CDC43substrates (12). These observations suggest that CDC43/CAL1encodes the [3 subunit of a PGT I-type protein prenyltransferase. Experimentsusing polypeptide substrates containing CaaL sequences demonstrate that CDC43/CAL1is indeed required for PGTI activity (40). Twoyeast genes may encode PGTIct subunits. The observation that mammalianPFT and PGTI contain a shared o~ subunit suggested that RAM2 mightbc the tx subunit for yeast PGTI, and the observation that coexpression of CDC43and RAM2in bacterial cells results in the production of active PGT I enzymesupports this hypothesis (J. B. Gibbs, personal communication). However,the ram2-1 point mutation caused only a twofold decrease in PGT I activity in vitro (40). This maysuggest that multiple yeast genes encode PGTot subunits. Interestingly, a second gene, knownas MAD2,has been isolated with sequence similarity to rat PFTet and RAM2.mad2 gain-of-function mutations werefirst identified on the basis of a defect in the regulation of the G2/Mtransition (81). Loss-of-function mutations in this gene cause arrest at G2/M,a phenotypesimilar to that of call mutants (R. Li, personal communication). Furthermore, the MAD2protein sequence contains calcium- binding motifs. These genetic data suggest that CDC43/CAL1may form heterodimers with both RAM2and MAD2.Thus, a subunits, like [3 subunits, may be shared between protein prenyltransferases. The CDC43-RAM2 heterodimermay prenylate proteins involved in cytokinesis and cell polarity, such as CDC42, whereas CDC43-MAD2may prenylate proteins such as RHO1and RHO2that are involved in the control of passage through G2/M. The cdc43 and call point mutations mayspecifically affect interaction of the CDC43protein with RAM2or MAD2,respectively. Intriguingly, these data also imply that the activity of the CDC43-MAD2enzyme may be regulated in a cell cycle-specific mannerby intracellular calcium signals. A third gene that encodes a protein with similarity to [3 subunits, known

by Medical Research Council-Cambridge UK on 11/21/06. For personal use only. as BET2,was identified on the basis of mutations that affect the secretory Annu. Rev. Genet. 1992.26:209-237. Downloaded from arjournals.annualreviews.org pathway. Since these mutations interact genetically with mutations in YPT1 and SEC4, both of which contain the carboxyl-terminal sequence C-C, BET2 is thought to encodethe [3 subunit of the yeast PGTII (119). bet2 mutations result in a defect in the membranelocalization of both YPT1and SEC4 proteins. In addition, cell extracts of bet2 mutants lack PGTII activity measured using bacterially expressed YPT1precursor (98). No promising candidates for a PGTII ot subunit have been identified, ram2mutants display no detectable defect in PGTII activity (98), and no genetic interactions have been observed between mad2and bet2 mutations (R. Li, personal communication). Thus, the ot subunit for this enzymemay be encoded by an as yet Annual Reviews www.annualreviews.org/aronline

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unidentified gene. Alternatively, the inability of carboxyl-terminal peptides to competefor PGTII activity in vitro suggests that PGTII maydiffer in reaction mechanism, and perhaps subunit structure, from PFTand PGTI. Preliminary evidence is consistent with a heterotrimeric structure for the Rab3APGT II (60). Therefore, the putative other subunit(s) of PGTII might not be homologousto previously characterized ~x subunits. Some prenylated proteins may be processed by multiple protein prenyltransferases. For example, yeast RAS2protein, which is primarily farnesylated by the RAM1-RAM2farnesyltransferase, also appears to be modifiedto a lesser, degree by a secondprenyltransferase activity. Yeast cells carrying a deletion of RAM1are viable, and a small but detectable amount of RAS2protein in these strains is in the membrane-associated,mature form (125). In contrast, a deletion of RAM2is lethal, and no membrane-associated RAS2is detected in ram2mutant strains (57). These data suggest that RAM1-independent, RAM2-dependentactivity is capable of weakly prenylat- ing RAS2in vivo. The [3 subunit of this enzyme may be CDC43,since overexpression of CDC43can suppress the growth defect of a raml mutant. In addition, raml cdc43 double mutants are inviable, but can be rescued by overexpression of RAS2(C. Trueblood & J. Rine, unpublished data). Since RAM2and CDC43appear to comprise a PGTI enzyme, some RAS2protein maybe geranylgeranylated in vivo; determination of the nature of the prenyl moiety of RAS2in ram1strains should address this possibility. Takentogether, these genetic data suggest a modelin whichthe [3 subunits of prenyltransferases confer protein substrate specificity, whereaset subunits provide catalytic function. This modelis consistent with biochemicalevidence that PFTot, but not PFT[3,binds the peptide substrate (113). Thus, sharing I~ subunits mayserve to generate divergent prenyltransferase specificities, whereas sharing of o~ subunits may generate multiple prenyltransferase isozymeswith commonspecificity but divergent regulatory properties.

Proteases by Medical Research Council-Cambridge UK on 11/21/06. For personal use only.

Annu. Rev. Genet. 1992.26:209-237. Downloaded from arjournals.annualreviews.org The maturation of manyprenylated proteins involves specific proteolytic processing. In particular, farnesylated and geranylgeranylated proteins with carboxyl-terminal CaaXsequences are further modified by the proteolytic removal of the three carboxyl-terminal amino acids, a modification necessary for subsequent carboxymethylation. A carboxyl-terminal protease activity active on Ras precursors is present in mammaliancell membranes(8, 52). Thus, whereasfamesylation of Ras precursors occurs in the cytosol, proteolysis most likely occurs at a membraneorganelle, perhaps the plasma membrane. The carboxyl-terminal protease is an endopeptidase (8), and requires a farnesylated substrate. The residue one-amino acid from the carboxyl-terminus(i.e. the a2 residue of the CalazXsequence) appears to Annual Reviews www.annualreviews.org/aronline

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important for the specificity of the mammaliancarboxyl-terminal protease, as mutations in this residue can prevent proteolysis, but not farnesylation, of K-ras in vivo (71). Yeast extracts also contain a membrane-associated carboxyl-terminal protease activity (8, 62). Like the mammalianenzyme, this enzymeis an endopeptidase that acts only on prenylated substrates. The membrane-associated protease is not encoded by any knowngene required for a-factor or RAS2processing. Geranylgeranylated peptides are also proteolyzedby a membrane-associatedprotease; it is presently unclear whether this activity is distinct from the famesyl-dependentmembrane protease (8). In addition to this membraneprotease, a carboxyl-terminal protease active on a-factor is present in the soluble fraction of a yeast extract (8, 62), and has been partially purified (62). This soluble enzymeis a processive exopeptidase that is not farnesyl-dependent. In fact, this enzymecan remove the farnesylated cysteine itself unless it is further modifiedby carboxymethylation. Therefore, it is not clear that this enzymeis relevant to the processing of prenylated proteins in vivo. The maturation of lamin A, a-factor, and M-matingpheromone of S. pombe also involves a second type of proteolytic processing. These proteins require endoproteases that cleave the polypeptide precursor at a site amino-terminal to the prenylated cysteine residue, resulting in the release of a prenylated oligopeptide. In vivo, the amino-terminal proteases involved in lamin A and a-factor maturation are active only on farnesylated precursors (11, 124). Presently, little is knownabout these enzymes. A yeast gene, STE19, has been identified that is required for the production of active a-factor, and is not deficient in the farnesyltransferase, carboxyl-terminalprotease, or methyltransferase activities (M. N. Ashby& J. Rine, unpublished). The amino-terminal proteolysis of a synthetic substrate based on a-factor can be catalyzed in vitro by a crude yeast extract (88). Usingthis assay, it should be possible to determine whether STE19encodes the amino-terminal protease enzyme. In addition to the proteases involved in the maturation of prenylated proteins, there are at least two examples of prenyl-dependent degradative

by Medical Research Council-Cambridge UK on 11/21/06. For personal use only. proteases. Strains of R. toruloides of the a-mating type produce an en- Annu. Rev. Genet. 1992.26:209-237. Downloaded from arjournals.annualreviews.org doprotease that cleaves the prenylated mating factor rhodotorucine A (95). This cleavage is necessary for metabolismof and response to rhodotorucine A by a cells. Similarly, S. cerevisiae strains of o~-matingtype producea cell surface-associated endoprotease that degrades a-factor (89). This protease inactivates the a-factor pheromonemolecule and promotes recovery from growth arrest. Both enzymes specifically degrade famesylated peptides; peptide analoguesthat lack the farnesyl group, or are modifiedwith a different alkyl group, are poor substrates for these enzymes. The sxal gene from S. pombe may encode a prenyl-dependent degradative protease; sxal mutants are hypersensitive to the M-factor lipopeptide pheromone(65). The possibility Annual Reviews www.annualreviews.org/aronline

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that prenyl-dependent degradative proteases mayplay a role in the turnover of prenylated proteins in other organisms remains unexplored. The primary sequence of the rhodotorucine A precursor suggests the existence of a protease that releases short oligopeptides, which are further processed to produce farnesylated pheromonemolecules (2). The details this process are unclear; it is not known,for example, whether proteolysis occurs before or after famesylation, or whetherthe earlier step is a precondition for the later step. At present, rhodotorucineA provides a novel example of prenyl modification of cysteine residues located in an internal region of the primary precursor. If the prenylation of internal cysteine residues is a general biological phenomenon,the details of this type of proteolytic maturation will acquire particular biological significance. Reversible Modifications The carboxyl-termini of prenylated proteins are often targets for additional modifications that, unlike proteolysis and prenylation, are reversible. In particular, palmitoylation has been implicated in the functions of several prenylated proteins, including some Ras, Rap, and Rab proteins. Among prenylated proteins, palmitoylation has been studied most extensively for the humanRas proteins. The palmitoyl modification of these proteins has a faster turnover time than the proteins themselves; thus, Ras proteins appear to be palmitoylated and depalmitoylated in a dynamicfashion (50). A palmitoyltransferase activity has been identified in mammaliancell extracts that can palmitoylate cytosolic N-ras precursors, using palmitoyl-CoA as a substrate (49). This activity is associated with Golgi-rich membrane fractions. Bacterially expressed Ras precursors are poor substrates, implying that the palmitoyltransferase mayrequire a farnesylated substrate. Since the mature N-ras protein is associated with the plasma membrane,and palmitoylation is dynamic, the apparent lack of palmitoyltransferase activity in the plasma membraneis perhaps surprising. Possibly this palmitoyltransferase activity maynot be relevant to Ras processing in vivo. It is interesting to

by Medical Research Council-Cambridge UK on 11/21/06. For personal use only. note, however, that yeast YPT1is palmitoylated and localized to the Golgi Annu. Rev. Genet. 1992.26:209-237. Downloaded from arjournals.annualreviews.org (97, 128). It is possible, therefore, that this palmitoyltransferaseis actually involved in palmitoylation of Rab/YPTproteins. Clearly, mutants in the palmitoyltransferase gene would be invaluable in identifying the relevant targets of this enzyme. Consideration of the mechanismof the palmitoylation of rhodopsin, an integral membraneprotein, mayprovide insight into the mechanismof Ras palmitoylation. Rhodopsinis posttranslationally modifiedby a thioester-linked palmitoyl moiety at the rod outer segment, an organelle derived from the plasma membrane(104). Palmitoylation of rhodopsin, like Ras, appears be dynamic. Surprisingly, the palmitoylation of rhodopsin mayoccur by a Annual Reviews www.annualreviews.org/aronline

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nonenzymatic mechanism(103). Although rod outer segments can transfer palmitate from palmitoyl-CoAto rhodopsin, this activity is insensitive to boiling, and copurifies with rhodopsinitself. Energetically, it is possible that both the transfer of a palmitoyl moiety from palmitoyl-CoAto cysteine and the subsequent hydrolysis of the cysteine thioester maynot require an enzyme for catalysis. However,since palmitoyl is the only fatty acyl moiety that is transferred to rhodopsin, somemechanism must exist to provide this specificity. Precedent exists for specific nonenzymaticmodification of proteins; for example, the maturation of rhodopsin involves the nonenzymaticcovalent attachment of a specific carotenoid molecule, ll-cis-retinal, to a specific amino acid of the opsin polypeptide (145). The parallels betweenpalmitoyla- tion of rhodopsin and Ras suggest that Ras may becomepalmitoylated and depalmitoylated at the plasma membraneby a similar nonenzymatic mechanism. A second modification commonto manyprenylated proteins is methylation. For example, Ras proteins, B-type lamins, some trimeric G-proteins, and some Rab proteins contain methylated carboxyl-termini. Whereas for Ras proteins the half-life of this modificationis identical to that of the protein itself (50), for several other prenyiated proteins, such as lamin B, the methyl group is turned over more rapidly (25), suggesting that carboxymethylation can be reversible. Reversible methylation of prenylated carboxyl-termini would presumably require the existence of a methyltransferase and a methylesterase enzyme. In yeast, a membrane-associatedmethyltransferase has been identified that is encodedby a single gene, STE14(61, 63). STE14 encodesan integral membraneprotein required for the methylation of a-factor and RAS2.Mutants defective in this gene are unable to produceactive a-factor, but display no other obvious phenotypes. Thus, methylation mayhave little importance for Ras function, at least in yeast. It is worth noting, however, that proteins such as lamin B and "C-X-C" Rab proteins, for which methylation may be a reversible, regulatory modification, have not been identified in S. cerevisiae. Methyltransferaseactivity has also been detected by Medical Research Council-Cambridge UK on 11/21/06. For personal use only.

Annu. Rev. Genet. 1992.26:209-237. Downloaded from arjournals.annualreviews.org in crude membranefractions from mammaliancells (132), and in rod outer segment (ROS)membranes (107). Interestingly, ROSmembranes contain active methylesterase activity (107). Since methylation appears to play important role in the interaction of transducin with the activated form of rhodopsin, these two enzymesmay provide an important function in regulating visual signal transduction by controlling the methylationstate of proteins such as transducin, cGMPphosphodiesterase, and rhodopsin kinase. The phosphorylation of Raplb protein provides an intriguing example of a reversible modification mediating the effects of an intracellular second messenger on the biological properties of a prenylated protein. Raplb is phosphorylated on a specific carboxyl-terminal serine residue by cAMP-de- Annual Reviews www.annualreviews.org/aronline

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pendent protein kinase (A-kinase) (56). This modification is important efficient interaction between the carboxyl-terminus of Raplb and the GDP exchange factor GDS(131). GDSstimulates both the exchange of GTPfor GDPbound to Raplb and the dissociation of Raplb from intracellular membranes. Thus, phosphorylation of Raplb by A-kinase results in an increase in the GTP-boundversus the GDP-boundform of the protein, and transfer of the protein molecule from a membranelocation to the cytosol. Interestingly, Ras proteins from both yeast and humansare phosphorylated on specific serine residues; in humanN-Ras, the site of phosphorylation has been localized to the carboxyl-terminus (27, 121). In RASproteins yeast, only the membrane-boundform is phosphorylated. Perhaps phosphorylation also regulates the interaction of Ras proteins with guaninenucleotide exchangefactors.

PRENYL-MEDIATED INTERACTIONS

Although considerable progress has been maderecently in identifying the enzymesthat direct protein prenylation and their protein targets, the waysin which prenyl modifications function remain largely unknown.In particular, although a primary function of prenylation for manymodified proteins is to direct intracellular localization, the mechanismsof these targeting processes, and the cellular factors required for them, have not been determined. Based on recent data (Figure 1), however, it is possible to speculate upon how prenyl-dependentprotein targeting might occur. At least three general types of modelsfor prenyl-mediated protein targeting can be proposed.

Prenyl-protein Receptors Prenylated carboxyl-termini could be directed to intracellular compartments by specifically localized membrane-boundreceptors. This type of mechanism is likely to be responsible for the plasmamembrane localization of myristoylated Src proteins (115). The putative myristoyl-Src receptor appears by Medical Research Council-Cambridge UK on 11/21/06. For personal use only.

Annu. Rev. Genet. 1992.26:209-237. Downloaded from arjournals.annualreviews.org recognize both the lipid moiety and the amino acids in the protein amino-terminus (116). Similarly, hypothetical prenyl-protein receptors would most likely recognize both the prenyl moiety and somespecific aminoacid sequence or protein structure, since proteins with identical lipid modifications can be localized to different subcellular compartments. The principle of interaction betweenprenylated polypeptides and specific receptors has been clearly established by work on fungal mating factors and their receptors. The prenylated mating pheromonea-factor interacts with a specific transmembranereceptor, STE3,to induce growth arrest and morpho- logical differentiation in cells of the ot mating type (51). Furthermore, experiments using synthetic analogues of a-factor have demonstratedthat the Annual Reviews www.annualreviews.org/aronline

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Figure1 Modelsfor prenyl-dependentprotein targeting. A. Prenyl-proteinreceptor model. A membrane-localizedreceptor specifically recognizes lipid andprotein structure. B. Dumblipid model.The lipid grouppromotes nonspecific affinity for membranes;specific targeting is mediated by protein-proteininteractions. C. Smartlipid model.Specific lipid-lipid interactions mediate proteinlocalization; bars indicate boundaries of a membraneregion of specificlipid composition. D. Secondaryinteraction model. A prenyl-dependent secondary modification mediates protein targeting.

receptor is highly specific for both the peptide sequenceand the posttranslational lipid modifications. Peptide analogues lacking an alkylated cysteine are essentially inactive. If the famesyl group is replaced with a different thioether-linked alkyl group, the activity of the resulting peptide is reduced; some analogues, such as S-geranyl a-factor, retain as muchas 50%of the activity of authentic a-factor, whereasS-methyl a-factor is only 0.2%as active as authentic a-factor in someassays (5, 87). Methylation is also important for the activity of a-factor; unmethylateda-factor analogues are generally inactive in inducinga-factor arrest (87). a-factor is inactivated by proteolysis (89); therefore, the peptide must also be important for recognition by STE3. The effects of the pheromone rhodotorucine A of R. toruloides and the

by Medical Research Council-Cambridge UK on 11/21/06. For personal use only. pheromonetremerogen A-10 of T. mesenterica, whoseactivities are also likely Annu. Rev. Genet. 1992.26:209-237. Downloaded from arjournals.annualreviews.org to be receptor-mediated, also require both the peptide sequenceand the lipid modificationfor biological activity (136). Interactions between the prenylated carboxyl-terminus of a-factor and a specifically localized transmembraneprotein may also be involved in the extracellular secretion of the pheromone. The secretion of farnesylated a-factor is knownto require the activity of a transmembraneglycoprotein, STE6.STE6 was identified on the basis of mutations that rendered yeast cells of the a-mating type mating-deficient (118). ste6 mutants are capable of synthesizing mature a-factor; however,this a-factor is not secreted (79). The sequence of STE6predicts that it encodes a membrane-spanningprotein with Annual Reviews www.annualreviews.org/aronline

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significant sequence similarity to bacterial membranetransporters and, in particular, mammalianP-glycoproteins (78, 92). Thus, STE6 is likely to encode a protein that pumpsprocessed a-factor across the plasma membrane. It is not knownwhether STE6protein specifically recognizes the farnesyl group of a-factor. However, since mutations that block famesylation of a-factor prevent its secretion by STE6, whereas a mutation that blocks methylation does not (90), either farnesylation or one of the proteolytic modifications must be essential for export by STE6. Mammaliancells contain genes that are highly homologousto STE6, the MDR(multidrug resistance) or P-glycoprotein genes. Overexpression of the MDR1gene in transformed cells results in resistance to a wide range of structurally divergent cytotoxic compounds,including vinblastin, vincristine, actinomycin, colchicine, and adriamycin (3, 28). This phenotyperesults from an increased efflux of drug from the treated cells, implying that the MDR1 protein is pumpingtoxins out of the cell (45). Other MDR1homologues are involved in translocation of MHCpeptides into the ER lumen (36). One might also speculate that an MDR-likeprotein might mediate the apparent prenyl- dependent entry of tremerogen A-10 precursor into the secretory pathway. Although the idea of prenyl-mediated intracellular trafficking mediated by MDRhomologues is reasonable, it is important to recognize that no MDR homologue, including STE6, has been directly demonstrated to require a prenyl modification for membranetranslocation at a peptide substrate. Another candidate for a prenyl-protein receptor that mediates protein targeting is the lamin B receptor, a 58-kd nuclear membraneprotein. This receptor mediates the association of lamin B with the nuclear membrane,a process that also depends on farnesylation (141). The gene for the lamin receptor, which has been cloned from chicken, is predicted to encode a polytopic integral membraneprotein (140). However,it is presently unknown whetherrecognition by the lamin B receptor specifically requires the farnesyl modification.

Lipid Interactions by Medical Research Council-Cambridge UK on 11/21/06. For personal use only. Annu. Rev. Genet. 1992.26:209-237. Downloaded from arjournals.annualreviews.org A second possible mechanism for prenyl-mediated protein targeting is association of prenylated proteins with cellular membranesby interactions between the prenyl group and membranelipids. Modelsof this type include the "dumb lipid" model, in which the prenyl group merely provides a nonspecific lipophilic glue that provides affinity for membranes,but does not mediate targeting to a specific membraneorganelle, and the "smart lipid" model,in whichthe lipid modificationdirects the protein to a specific locale through a nonreceptor-based mechanism. A type of smart lipid model has been proposed to explain the localization of GPI-linked proteins to plasma- membranesites called caveolae (6). Clustering of GPI-linked proteins, such Annual Reviews www.annualreviews.org/aronline

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as the folate receptor in caveolae, is disrupted by drugs, such as filipin and nystatin, that bind cholesterol (120). Thus, it has been suggested that the specific lipid composition of caveolae attracts GPI-linked proteins through specific lipid interactions. A lipid interaction modelis consistent with muchof the information on the localization ofprenylated Ras-superfamilyproteins. Clearly, farnesylation and the other carboxyl-terminalmodifications serve to increase the affinity of Ras proteins for membranes.For K-ras proteins synthesized in vitro, famesylation, proteolysis, and methylation all have the cumulative effect of increasing the membraneaffinity of the Ras protein (52). Similarly, the membraneaffinities of mutant H-ras proteins lacking palmitoylation sites and K-ras proteins lacking a polybasic region are markedly less than wild-type forms of these proteins (53). Some experiments with mutant Ras proteins suggest that prenylation is a nonspecific membrane-localizationsignal. For example, K-ras mutant proteins with a CaaL sequence identical to Rapla are geranylgeranylated rather than farnesylated, yet are still localized to the plasm~ membranerather than the Golgi (53). Similarly, the biological activity Rapl a, a Golgi-associatedprotein that suppresses Ras activation, is unaffected by replacing the geranylgeranyl modification with farnesyl (30). In addition, the yeast YPT1protein, a member of the Rab family that contains the geranylgeranylation sequence C-Cat its carboxyl-terminus, retains biological activity when this sequence is altered to produce a CaaXfarnesylation sequence(97). Theseresults suggest that targeting of these proteins to specific membranecompartments does not involve the prenyl group, but rather the amino acid sequence of the proteins. The carboxyl-termini of small GTP-bindingproteins contain a hypervariable region that may direct endomembranetargeting. Mutations that affect the polybasic region of the hypervariable domain of geranylgeranylated K-ras do not affect membraneaffinity in vitro, nor membranepartitioning in vivo. However,these mutant proteins are targeted neither to the plasma membrane, like K-ras, nor the Golgi, like Rapl a, but to manymembranes in an apparently

by Medical Research Council-Cambridge UK on 11/21/06. For personal use only. nonspecific manner (53). The hypervariable domain of geranylgeranylated Annu. Rev. Genet. 1992.26:209-237. Downloaded from arjournals.annualreviews.org Rabproteins also appears to be critical to the localization of these proteins to specific membranecompartments (24). Thus, in K-ras, Rapl, and at least some Rab proteins, prenylation, proteolysis, and methylation appear to promotenonspecific membraneassociation, whereas the hypervariable region directs localization to specific membranes. Althoughthe above results suggest that the farnesyl modification of K-ras is a dumblipid, experiments with H-ras suggest that the prenyl moiety may provide morethan nonspecific lipophilicity. Mutationof the carboxyl-terminal residue of H-ras to leucine (30, 53), or deletion of the CaaXsequence and insertion of an amino-terminal myristoylation sequence (20), results in the Annual Reviews www.annualreviews.org/aronline

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production of mutant H-ras proteins that are either geranylgeranylated or myristoylated, rather than famesylated. Both mutant proteins are membrane associated; for the geranylgeranylated form, the site of localization is the plasma membrane(53). In addition, activated forms of these proteins were capable of inducing cellular transformation. However,the nonactivated forms of these proteins behaveddifferently from farnesylated H-ras; geranylgeranylated H-ras exhibits a dominant negative phenotype (30), and myristoylated H-ras has a transforming phenotype(20). Interestingly, H-ras, but not K-ras, is palmitoylated. Perhaps the interactions between the farnesyl group, the palmitoyl group, and the membranelipids serve to orient the protein in a way that is important for wild-type, but not transforming,function. In this regard, note that palmitoylation has been suggested to be important for orienting rhodopsin in the rod outer segment membrane(103).

Indirect Prenyl-dependent Targeting prenylation itself need not play a direct role in mediating protein targeting; in some cases, prenylation mayinstead be a precondition for a secondary modificationthat actually directs protein localization. The processingof lamin A provides a clear case of an indirect effect of prenylation on protein localization. Famesylationof prelamin A is clearly required for the assembly of normal lamin A protein into the nuclear lamina, since inhibition of this process results in accumulation of prelamin A in nuclear inclusions (11). However,famesylation per se is not required for this process. Rather, the essential step is removalof the prelamin A carboxyl-terminus,which, in turn, requires farnesylation, as lamin A precursors lacking the carboxyl-terminal amino acids are assembled into the lamina in the complete absence of prenylation (82). Prenyl-dependentproteolysis mayalso be essential for the secretion of a-factor of yeast. Althoughthe requirements for STE6-mediated export of a-factor have not been determined, it is possible that the critical processing step required for this process is proteolysis of the amino-terminal pro region of the a-factor precursor. Reversible prenyl-dependent modifications may be important for the by Medical Research Council-Cambridge UK on 11/21/06. For personal use only.

Annu. Rev. Genet. 1992.26:209-237. Downloaded from arjournals.annualreviews.org localization and function of prenylated proteins. For example, methylation appears to be required for translocation of transducin ~’~ from the cytosol to the ROSmembrane, where it interacts with transducin tx and rhodopsin (105). Activation of rhodopsin with light stimulates the membranelocalization of methylatedtransducin 13~/, a stimulation that requires transducin or. Thus, the effect of methylation on the membrane-targetingof transducin 13~/appears to result, at least in part, from enhancedprotein-protein interactions between transducin ~x and 13~/. The existence of several proteins involved in visual signal transduction, including rhodopsin kinase and cGMPphosphodiesterase, that are similarly farnesylated and carboxymethylated,suggests that reversible Annual Reviews www.annualreviews.org/aronline

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methylation maybe critical for the interaction of all these proteins with rhodopsin and with one another. The example of transducin also suggests a similar role for methylation and palmitoylation in the cycling of Raband Rho proteins betweenthe cytosol and cellular membranes;this possibility remains to be explored. These models for prenyl-dependent protein targeting are not mutually exclusive. For example, the interaction of geranylgeranylated Raplb with its GDSrequires the geranylgeranyl group, the carboxyl-terminal peptide sequence, and the phosphorylatedserine residue. This interaction is correlated with transit of Raplb from the Golgi membraneto the cytosol (56). Thus, this process involves a protein-prenylinteraction that is analogousto the prenyl receptor model, yet also requires a secondary modification. The targeting of manyprenylated proteins may likewise involve mechanisms that contain elements of several of the models described here.

FUTURE PERSPECTIVES

Althoughmuch has been learned about protein prenylation in a relatively short period of time, manycritical questions remain unanswered. Clearly, the precise functional role played by the prenyl group in protein localization and function is only beginning to be understood, and understanding this role is therefore a majorfocus of current research. In addition, the identification and characterization of new protein prenyltransferases and other enzymesinvolved in prenyl-dependentmodifications remains an important area of research, since the existence of newenzymes with novel substrate specificities appears likely. In particular, the combinatorial sharing of protein prenyltransferase subunits could provide both diverse protein and lipid specificities and differential regulation of protein prenylation. Theidentification of newprenylated proteins may also provide conceptual breakthroughs, particularly if new prenyl modifications or newmodification sites are identified. Understanding protein prenylation mayprovide insight into two related

by Medical Research Council-Cambridge UK on 11/21/06. For personal use only. biological phenomenaof medical significance: oncogenesis, and cholesterol Annu. Rev. Genet. 1992.26:209-237. Downloaded from arjournals.annualreviews.org metabolism. The Ras proteins are well knownfor their oncogenic potential; activated Ras genes have been found in a variety of types of humantumors, including colorectal tumors, lung adenocarcinomas, and pancreatic tumors (10). Prenylation has been shownto be important for the functions of Ras proteins, since inhibition of prenylation suppresses the phenotypesof consti- tutively active Ras proteins in yeast (109). Furthermore, genetic and pharmacological inhibition of Ras processing in animal cells likewise suppresses Ras-induced transformation (31, 64). Thus, a greater understanding of the catalytic mechanismof protein prenyltransferases mayaid in the design of PFTinhibitors with potential utility for cancer treatment. Inhibitors of isoprenoid biosynthesis mayalso be useful for cancer therapy; several studies Annual Reviews www.annualreviews.org/aronline

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have indicated that inhibitors of mevalonate synthesis suppress Ras-induced cell transformation (110, 126, 33). Protein prenylation may also be important for the regulation of HMG-CoA reductase (HMGR),the rate-limiting step in cholesterol biosynthesis. The HMGRinhibitor mevinolin is commonly used to treat hypercholesterolemia, since mevinolin treatment results in increased synthesis of the LDLreceptor (17, 83). However, depletion of nonsterol isoprenoids also causes an increase in the translation of the HMGRmessage and a decrease in HMGRdegradation, resulting in abnormally high levels of HMG-CoAreductase protein (16, 101). Thus, in patients undergoing treatment with mevinolin, the cholesterol biosynthetic potential is actually elevated. Understanding the mechanism of translational and half-life control may provide insights into how this increase in HMGRsynthesis could be prevented. Prenylated proteins could mediate both processes, although other models involving mevalonate-binding proteins or prenylated mRNAare also plausible. Further studies should clarify the mechanism of translational regulation of HMGR,and perhaps provide practical insights into more effective treatment of hypercholesteremia.

ACKNOWLEDGMENTS We thank the many colleagues who provided reprints, recent preprints, unpublished observations, and stimulating discussions, and regret omissions demanded by space limitations. In addition, we thank the members of our laboratory for many suggestions and insights. Workin the authors’ laboratory was supported by grants from the National Institutes of Health (GM35827), the California Tobacco-related Disease Research Program, and by a postdoc- toral fellowship from Merck, Inc.

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