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PROTEIN PRENYLATION:
GENES, ENZYMES, TARGETS,
AND FUNCTIONS
William
R. Schafer and Jasper Rine
Departmentof Molecular and Cell Biology, University of California,
California 94720
Berkeley,
KEYWORDS:
Prenylation, posttranslational modification, isoprenoid, Ras proteins, palmitoylation
CONTENTS
INTRODUCTION ..........................................
GENES ENCODING PRENYLATED PROTEINS ......................
The Ras Superfamily
......................................
Lipopeptide
Pheromones ....................................
Nuclear
Lamins .........................................
G-proteins
............................................
PROTEINS INVOLVED IN PRENYL-DEPENDENTPROCESSING ..........
Protein Prenyltransferases
...................................
Proteases
.............................................
Reversible
Modifications
....................................
PRENYL-MEDIATED INTERACTIONS ...........................
Prenyl-protein
Receptors
....................................
Lipid Interactions
........................................
Indirect Prenyl-dependent
Targeting ............................
FUTURE PERSPECTIVES ....................................
209
210
210
213
215
216
217
218
221
223
225
225
227
229
230
INTRODUCTION
Theregulation,functionalactivation, andintracellular targetingof biological
macromoleculesare often mediated through the covalent attachment of
specific chemicalmoieties. The posttranslational modificationof specific
proteins by phosphorylation,glycosylation, acetylation, fatty acylation, and
methylationhas beenstudied extensively, anda variety of critical biological
functions have beenascribed to these particular side groups. In contrast to
these familiar types of chemicalmodification,protein prenylationis a process
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SCHAFER& RINE
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 oncogenesisand
hypercholesterolemia.
Prenylation refers to the covalent modification of a molecule by the
attachment of a lipophilic isoprenoid group (reviewed in 26, 29, 46). Isoprenoids 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 compoundsmost 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 (coenzymeQ), 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 attachmentof either
a 15-carbonfarnesyl or a 20-carbon geranylgeranylgroup (117) in a thioether
linkage to a cysteine residue. In most organisms, geranylgeranylation is a
more common
modification than farnesylation, although the relative proportions of farnesylcysteine and geranylgeranylcysteinein total cellular protein
vary somewhatbetweenorganismsand 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,
including the nucleus, the cytosol, and membrane-bound
organelles (85, 129).
GENES ENCODINGPRENYLATEDPROTEINS
Mostof the prenylated proteins whoseidentities have been determinedbelong
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
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PROTEINPRENYLATION 211
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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
MOTIFThe Ras proteins are plasma membrane-localized molecules that regulate cell differentiation and proliferation in mammalian
and
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
aminoacids 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 palmitoylatedbut 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 membrane
association 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
in mediating the association of Ras proteins with membranes.Whereashuman
Ras proteins lacking these modifications exhibit a reduced affinity for
membranes
(52), mutantK-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 additionto the farnesyl group,either a palmitoylationsite or a polybasic
region is required for efficient localization to the plasmamembraneand is
important for the transforming activity of oncogenicmutantproteins (55).
THECAALMOTIFA second group of small GTP-binding proteins contain a
carboxyl-terminal motif similar to a CaaXbox, called a CaaLmotif, with a
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SCHAFER& RINE
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 processingat 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
aminoacids; (c) methylation of the new carboxyl-terminus, and (d) phosphorylation of the distal serine residue by A-kinase (72). A prenylated carboxylterminus is required for the association of Raplbwith membranes.Prenylation
and phosphorylation are both required for interaction of Raplbwith a factor
knownas GDS,which stimulates GDP/GTP
exchange (56), since prenylated,
phosphorylated peptides corresponding to the Raplb carboxyl-terminus can
competewith intact Raplb for interaction with GDS,whereasother 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/GTP
exchange factors, the stimulatory factor GDSand the
inhibitory factor GDI.In contrast, bacterially expressed RhoAprotein, which
lacks carboxyl-terminalprocessing, is defective in all three of these interactions (59). Prenylation maybe generally involved in mediating interactions
betweenother small GTP-bindingproteins and exchangefactors (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 YPT5of Schizosaccharomyces
pombe,have been shownto be geranylgeranylated in vivo (73, 102); genetic
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PROTEIN PRENYLATION 213
and biochemical evidence indicates that others, including the mammalian
Rablb, Rab2, and Rab5 (75), and YPTI of S. cerevisiae (119), are
geranylgeranylatedas well. Usinga 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 carboxylterminal 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.
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 matingpheromone
a-factor of yeast. Yeast contain two a-factor genes, MFA1and MFA2,which
encode polypeptide precursors of 36 and 38 aminoacids, respectively, each
with a carboxyl-terminal CaaXsequence (15, 93). The a-factor precursor
undergoesextensive processing independentlyof the classic secretory pathway
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SCHAFER& RINE
before being secreted as the mature12-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) attachmentof 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 aminoterminus. 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), whereasunmethylateda-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 basidiomycetousjelly fungus Rhodosporidium
toruloides, is an 11-aminoacid peptide
with a carboxyl-terminal cysteine covalently boundto a farnesyl group in a
thioether linkage (69). In contrast to a-factor, the rhodotorucineA carboxylterminal cysteine is not methylated. Three genes, RHA1,RHA2and RHA3,
encode rhodotorucine A precursors consisting of three to five tandemcopies
of the rhodotorucine A peptide (2). These peptide sequences are separated
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)
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PROTEINPRENYLATION 215
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-10is synthesized as a large precursor of unknown
primary sequence. The secreted pheromoneis 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
undergothe 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 pheromonesmaybe ubiquitous amongthe fungi. It
unclear whether prenylated peptide hormonesare found in other organisms.
Nuclear
Lamins
Laminsare nuclear proteins that are a major componentof the karyoskeleton.
Laminsare 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
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SCHAFER& RINE
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 CaaXsequenceis 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 laminB, and
a precondition for other processing steps (11,137). Since the presence of the
CaaXsequence 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 beenproposed
(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
laminA 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).
G-proteins
G-proteins are heterotrimeric GTP-binding proteins that mediate signal
transduction between transmembranereceptors and intracellular second messengers. 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 membrane
localization and functional activity (41). A numberof uniden-
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PROTEINPRENYLATION 217
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 cGMPphosphodiesterase, 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, whereasthe [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).
Theinvolvementof several prenylated proteins in a. single signal transduction
pathwaysuggests 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
PROCESSING
IN
PRENYL-DEPENDENT
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 attachmentof 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 mediatingthe common
and divergent
biological properties of the processed carboxyl-termini.
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Protein
SCHAFER& RINE
Prenyltransferases
BIOCHEMICAL
STUDIES
Several enzymes, termed protein prenyltransferases,
which catalyze the attachmentof a specific prenyl moietyto specific protein
precursors, have been identified and characterized. At least three distinct
protein prenyltransferase activities are present in yeast and mammalian
cells.
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 secondactivity, termedprotein geranylgeranyltransferase I (PGT
has also been identified in soluble extracts. This enzyme,whichis 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 immunodeplete
PGTI activity from crude extracts, it
has been hypothesized that mammalianPGTI is also a heterodimer with an
ct subunit that is shared with PFT(127). A third enzyme,PGTII, 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).
Althoughboth 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,
whereasRas farnesylation occurs in the cytoplasm, maysuggest the existence
of a distinct nuclear farnesyltransferase (82). In this regard, it has been
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
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PROTEINPRENYLATION 219
this hypothesis, proteins containing S, C, M, Q, or A in this position are
substrates for PFT, whereasproteins 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 weaklyin vitro on the preferred
substrates of the other enzyme(144).
The PGTII enzymeshowsstalking substrate specificity. Intriguingly, the
sequences required for geranylgeranylation by PGTII, unlike PFTand PGT
I, are not exclusively located at the carboxyl-terminus(98). Geranylgeranylation by PGTII is not competedby peptides corresponding to the carboxyltermini 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.
GENETIC
STUDIES
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 RAM1
and RAM2
proteins,
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 RAM1
have been isolated
independently by several groups based on several phenotypes: suppression of
RAS2activation (43, 109), a defect in the production of maturea-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 havenot beenidentified in yeast cells).
Mutations in RAM2
also suppress RAS2activation and block production of
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
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SCHAFER& RINE
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/CAL1
encodes the [3 subunit of a PGT
I-type protein prenyltransferase. Experimentsusing polypeptide substrates
containing CaaL sequences demonstrate that CDC43/CAL1
is indeed required
for PGTI activity (40).
Twoyeast genes may encode PGTIct subunits. The observation that
mammalianPFTand PGTI contain a shared o~ subunit suggested that RAM2
mightbc the tx subunit for yeast PGTI, and the observation that coexpression
of CDC43
and RAM2
in 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.mad2gain-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 MAD2
protein sequence contains calciumbinding motifs. These genetic data suggest that CDC43/CAL1
may form
heterodimers with both RAM2
and MAD2.Thus, a subunits, like [3 subunits,
may be shared between protein prenyltransferases.
The CDC43-RAM2
heterodimermayprenylate proteins involved in cytokinesis and cell polarity,
such as CDC42, whereas CDC43-MAD2
may prenylate proteins such as
RHO1and RHO2that are involved in the control of passage through G2/M.
The cdc43and call point mutations mayspecifically affect interaction of the
CDC43protein with RAM2
or MAD2,respectively. Intriguingly, these data
also imply that the activity of the CDC43-MAD2
enzymemay be regulated
in a cell cycle-specific mannerby intracellular calcium signals.
A third gene that encodes a protein with similarity to [3 subunits, known
as BET2,was identified on the basis of mutations that affect the secretory
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 enzymemaybe encoded by an as yet
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PROTEIN PRENYLATION 221
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
Rab3APGTII (60). Therefore, the putative other subunit(s) of PGTII might
not be homologousto previously characterized ~x subunits.
Someprenylated proteins may be processed by multiple protein prenyltransferases. For example, yeast RAS2protein, which is primarily farnesylated by the RAM1-RAM2
farnesyltransferase,
also appears to be
modifiedto a lesser, degree by a secondprenyltransferase activity. Yeastcells
carrying a deletion of RAM1
are 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 RAM2
is lethal, and no membrane-associated
RAS2is detected in ram2mutant strains (57). These data suggest that
RAM
1-independent, RAM2-dependent
activity is capable of weakly prenylating RAS2in vivo. The [3 subunit of this enzyme may be CDC43,since
overexpression of CDC43
can 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
moietyof 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 common
specificity but divergent regulatory properties.
Proteases
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
removalof the three carboxyl-terminal aminoacids, a modification necessary
for subsequent carboxymethylation. A carboxyl-terminal protease activity
active on Ras precursors is present in mammalian
cell 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
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222
SCI-IAFER& RINE
important for the specificity of the mammalian
carboxyl-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 mammalian
enzyme,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-associated
protease; it is presently unclear whether
this activity is distinct from the famesyl-dependentmembrane
protease (8).
In addition to this membrane
protease, 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-matingpheromoneof S. pombe
also involves a second type of proteolytic processing. Theseproteins 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 examplesof prenyl-dependent degradative
proteases. Strains of R. toruloides of the a-mating type produce an endoprotease 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 enzymesspecifically 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
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PROTEINPRENYLATION 223
that prenyl-dependentdegradative 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
matureN-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
note, however, that yeast YPT1is palmitoylated and localized to the Golgi
(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
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SCHAFER& RINE
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, somemechanismmust 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
aminoacid of the opsin polypeptide (145). The parallels betweenpalmitoylation 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 membrane
protein required for the methylation of a-factor
andRAS2.Mutantsdefective in this gene are unable to produceactive a-factor,
but display no other obvious phenotypes. Thus, methylation mayhave little
importancefor 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
in crude membranefractions from mammaliancells (132), and in rod outer
segment (ROS)membranes(107). Interestingly, ROSmembranescontain
active methylesterase activity (107). Since methylation appears to play
important role in the interaction of transducin with the activated form of
rhodopsin, these two enzymesmayprovide an important function in regulating
visual signal transduction by controlling the methylationstate of proteins such
as transducin, cGMP
phosphodiesterase, and rhodopsin kinase.
The phosphorylation of Raplb protein provides an intriguing exampleof 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-
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PROTEINPRENYLATION 225
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-bound
form 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-bound
form is phosphorylated. Perhaps phosphorylation also regulates the interaction of Ras proteins with guaninenucleotide
exchangefactors.
PRENYL-MEDIATED
INTERACTIONS
Althoughconsiderable 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 mechanisms
of 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-bound
receptors. This type of mechanism
is likely to be responsible for the plasmamembrane
localization of myristoylated Src proteins (115). The putative myristoyl-Src receptor appears
recognize both the lipid moietyand the aminoacids in the protein amino-terminus (116). Similarly, hypothetical prenyl-protein receptors would most
likely recognize both the prenyl moietyand 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 morphological differentiation in cells of the ot mating type (51). Furthermore,
experimentsusing synthetic analogues of a-factor have demonstratedthat the
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SCHAFER& RINE
Figure1 Models
for prenyl-dependent
proteintargeting.A.Prenyl-protein
receptormodel.A
membrane-localized
receptorspecificallyrecognizes
lipid andproteinstructure.B. Dumb
lipid
model.
Thelipid grouppromotes
nonspecific
affinityfor membranes;
specifictargetingis mediated
byprotein-protein
interactions.C. Smartlipid model.Specificlipid-lipidinteractionsmediate
proteinlocalization;
barsindicateboundaries
of a membrane
regionof specificlipid composition.
D.Secondary
interactionmodel.Aprenyl-dependent
secondary
modification
mediatesprotein
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-methyla-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
pheromonetremerogenA-10of T. mesenterica, whoseactivities are also likely
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.STE6was identified on the basis of mutations that rendered yeast cells
of the a-mating type mating-deficient (118). ste6 mutants are capable of
synthesizing maturea-factor; however,this a-factor is not secreted (79). The
sequence of STE6predicts that it encodes a membrane-spanning
protein with
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PROTEIN PRENYLATION227
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. Overexpressionof 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 MDR1
homologuesare
involved in translocation of MHC
peptides into the ERlumen(36). One might
also speculate that an MDR-likeprotein might mediate the apparent prenyldependent entry of tremerogen A-10 precursor into the secretory pathway.
Althoughthe idea of prenyl-mediated intracellular trafficking mediated by
MDR
homologuesis reasonable, it is important to recognize that no MDR
homologue, including STE6, has been directly demonstrated to require a
prenyl modification for membrane
translocation 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 membrane
protein (140). However,it is presently unknown
whetherrecognition by the laminB receptor specifically requires the farnesyl
modification.
Lipid Interactions
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 plasmamembrane
sites called caveolae (6). Clustering of GPI-linked proteins, such
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SCHAFER& RINE
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-terminalmodificationsserve 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
membrane
affinity of the Ras protein (52). Similarly, the membrane
affinities
of mutant H-ras proteins lacking palmitoylation sites and K-ras proteins
lacking a polybasic region are markedlyless than wild-type forms of these
proteins (53). Someexperiments 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 suppressesRas activation, is unaffected
by replacing the geranylgeranyl modification with farnesyl (30). In addition,
the yeast YPT1protein, a memberof the Rab family that contains the
geranylgeranylationsequence 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
aminoacid sequence of the proteins.
The carboxyl-termini of small GTP-bindingproteins contain a hypervariable
region that may direct endomembrane
targeting. Mutations that affect the
polybasic region of the hypervariable domainof 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
nonspecific manner (53). The hypervariable domain of geranylgeranylated
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, whereasthe hypervariable region
directs localization to specific membranes.
Althoughthe aboveresults 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
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PROTEIN PRENYLATION 229
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 dominantnegative 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 membrane
lipids serve to orient the protein in a way
that is importantfor 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 mediatingprotein 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
localization and function of prenylated proteins. For example, methylation
appears to be required for translocation of transducin ~’~ from the cytosol to
the ROSmembrane,whereit interacts with transducin tx and rhodopsin(105).
Activation of rhodopsin with light stimulates the membrane
localization of
methylatedtransducin 13~/, a stimulation that requires transducin or. Thus, the
effect of methylation on the membrane-targetingof transducin 13~/appearsto
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 cGMP
phosphodiesterase,
that are similarly farnesylated and carboxymethylated,
suggests that reversible
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230
SCHAFER& RINE
methylation maybe critical for the interaction of all these proteins with
rhodopsin and with one another. The exampleof transducin also suggests a
similar role for methylationand 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 Raplbwith 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 processinvolvesa 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 modelsdescribed here.
FUTURE
PERSPECTIVES
Althoughmuchhas 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 newenzymeswith 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
biological phenomenaof medical significance: oncogenesis, and cholesterol
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 constitutively 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
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PROTEIN PRENYLATION
231
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-limitingstep in cholesterol biosynthesis. The
HMGR
inhibitor 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 HMGR
message and a decrease in HMGR
degradation,
resulting in abnormally high levels of HMG-CoA
reductase protein (16, 101).
Thus, in patients undergoing treatment with mevinolin, the cholesterol
biosynthetic potential is actually elevated. Understanding the mechanismof
translational and half-life control may provide insights into how this increase
in HMGR
synthesis could be prevented. Prenylated proteins could mediate
both processes, although other models involving mevalonate-binding proteins
or prenylated mRNA
are 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 manysuggestions 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 postdoctoral fellowship from Merck, Inc.
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