(Received for publication, September 10, 1996, and in revised form, November 27, 1996)
From the Department of Biochemistry and Molecular
Biology, James H. Quillen College of Medicine, East Tennessee State
University, Johnson City, Tennessee 37614-0581, § Department
of Physiology, University of Colorado Health Sciences Center,
Denver, Colorado 80262, ¶ Amgen Inc., Boulder, Colorado 80301, and
Amgen Inc., Thousand Oaks, California 91320-1789
The 72-kDa nuclear lamina protein lamin A is synthesized as a 74-kDa farnesylated precursor. Conversion of this precursor to mature lamin A appears to be mediated by a specific endoprotease. Prior studies of overexpressed wild-type and mutant lamin A proteins in cultured cells have indicated that the precursor possesses the typical carboxyl-terminal S-farnesylated, cysteine methyl ester and that farnesylation is required for endoproteolysis to occur. In this report, we describe the synthesis of an S-farnesyl, cysteinyl methyl ester peptide corresponding to the carboxyl-terminal 18 amino acid residues of human prelamin A. This peptide acts as a substrate for the prelamin A endoprotease in vitro, with cleavage of the synthetic peptide at the expected site between Tyr657 and Leu658. Endoproteolytic cleavage requires the S-prenylated cysteine methyl ester and, in agreement with transfection studies, is more active with the farnesylated than geranylgeranylated cysteinyl substrate. N-Acetyl farnesyl methyl cysteine is shown to be a noncompetitive inhibitor of the enzyme. Taken together, these observations suggest that there is a specific farnesyl binding site on the enzyme which is not at the active site.
Proteins with a CAAX consensus sequence at their
carboxyl terminus undergo serial post-translational modifications of
the cysteinyl residue (1, 2). These modifications include
derivitization of the cysteine sulfhydryl with an isoprenoid moiety
followed by the endoproteolytic removal of the -AAX
tripeptide and methylation of the cysteine -carboxyl group. When the
X amino acid is S, C, Q, or M, a 15-carbon farnesyl residue
is attached in thioether linkage to the cysteine (3), whereas when
X is a leucine, a 20-carbon geranylgeranyl residue is found
instead (4).
The nuclear lamina is a thin, fibrous structure that lines the inner nuclear membrane and is believed to function in maintaining nuclear shape and volume (5) and may also be involved in the organization of chromatin in the interphase nucleus (6). In most mammalian cells, it consists of three class V intermediate filament proteins, lamins A, B, and C (5, 6). Prelamin A is the 74-kDa precursor of the 72-kDa nuclear lamin A protein (7). It possesses a CAAX box sequence (CSIM) (8, 9) and has been shown to be farnesylated in vitro (10) and in vivo (11). Despite the loss of the carboxyl-terminal 18 amino acids of prelamin A in its proteolytic conversion to lamin A, it nevertheless undergoes all of the reactions characteristic of other CAAX proteins (11). Experiments with mutants, in which the cysteine of the CAAX box is replaced by another amino acid, demonstrate that farnesylation is required for the maturation of prelamin A (12). These nonprenylated CAAX box mutants of prelamin A enter the nucleus, yet are not proteolytically processed and are not incorporated into the nuclear lamina. Similar results have been obtained with nonprenylated prelamin A produced by treating cultured mammalian cells with mevinolin (13, 14) or inhibitors of protein farnesylation (15).
Prelamin A is quantitatively converted to mature lamin A in mammalian
cell nuclei, consistent with a direct precursor-product relationship
and, hence, with a second endoproteolytic cleavage after the canonical
CAAX box modifications (13). Based on a comparison of the
predicted sequence for human prelamin A from its cDNA, and direct
sequencing of the carboxyl terminus of the mature lamin A molecule,
this second endoproteolysis is expected to be between a tyrosine
(Tyr657) and a leucine (Leu658) 18 amino acid
residues upstream from the carboxyl terminus of the prelamin A molecule
(16). Consistent with this expectation, mutation of Leu658
to arginine prevents conversion of prelamin A to mature lamin A (17).
These observations argue against the sequential action of multiple
proteolytic cleavages in conversion of the methylated and farnesylated
intermediate to mature lamin A. Rather, they support the hypothesis
that there is a single endoprotease that cleaves this intermediate
between Tyr657 and Leu658. We refer to this
activity as the "prelamin A endoprotease." A schematic diagram of
the prelamin A processing pathway concluding with the reaction
catalyzed by the prelamin A endoprotease is shown in Fig.
1.
In this report, we describe a cell-free assay for the prelamin A endoprotease and use this assay to characterize its specificity for various substrates. The results indicate that the prelamin A endoprotease has a specific binding site for the farnesyl group and, therefore, is somewhat analogous to the previously described isoprenylated protein "-AAX" endoprotease (18, 19), whose activity is also shown in Fig. 1. It will be seen that these two enzymes differ significantly, however, in that the prelamin A endoprotease is competitively inhibited by nonprenylated peptides, whereas the isoprenylated protein endoprotease is not (18).
HeLa cells were cultured in Ham's F-12 medium supplemented with 10% fetal calf serum (v/v) 100 units/ml penicillin, 100 µg/ml streptomycin, and 1 µg/ml amphotericin B. Chinese hamster ovary (CHO)-K1 cells with an up-regulated carboxylate transporter, Met-18b-2 (20, 21), were cultured in Ham's F-12 medium supplemented with 5% fetal calf serum plus antibiotics as for the HeLa cells.
Labeling of cells with [3H]mevalonate and
[35S]methionine, radioimmunoprecipitation of prelamin A,
and mature lamin A and treatment of cells with the farnesyl protein
transferase inhibitor, BZA-5B, have all been described elsewhere (15).
As in the previous study, prelamin A was immunoprecipitated with the
human species specific prelamin A antibody -PA (14), whereas total
lamin A was immunoprecipitated with a panspecies-specific lamin A
antibody (a kind gift of Dr. Nilabh Chaudhary, Triplex
Pharmaceuticals).
The polypeptide
H2N-RSYLLGNSSPRTQSPQNCOCH3 (prelamin A peptide) was
synthesized by stepwise solid phase,
Fmoc1/tert-butyl-based chemistry. An ABI-431-A
synthesizer (Perkin-Elmer) programmed with the manufacturer's standard
single coupling protocol was used to assemble the sequence. The
synthesis was initiated with Fmoc-Cyss(Trt)-Wang derivatized
polystyrene resin, and all subsequent couplings were carried out with
preformed HOBt esters. Following removal of the N-terminal Fmoc group,
the peptide resin was cleaved and deprotected by treatment with a
mixture of trifluoroacetic acid:thioanisole:-mercaptoethanol:water:phenol (80:5:5:5) for 4 h. The suspension was filtered and the resulting filtrate was concentrated under reduced pressure. The crude ether precipitate was
applied to a preparative Vydac C-4 column and eluted with a linear
0.1% trifluoroacetic acid, acetonitrile gradient system, and the
fractions with the best analytical profile were pooled and lyophilized.
Conversion to the methyl ester was accomplished in 50% yield by
stirring the free peptide in a 5% HCl, methanol solution for 4 h
at room temperature. The product was then isolated by utilizing the
same HPLC conditions as for the free peptide. Results
of amino acid and electrospray mass spectrometry analyses corresponded
closely with expected values.
The S-all-trans-farnesyl peptide derivative was synthesized (22) by a base catalyzed reaction with farnesyl bromide (Aldrich). The prelamin A peptide (4.33 µmol) was dissolved in 15 ml of dimethylformamide, H2O, 0.5 M KHCO3 (5:1:1), and then 6.5 µmol of farnesyl bromide were added in 1.85 ml of dimethylformamide. The reaction was in the dark at room temperature for 20 min and was terminated by the addition of 0.67 ml of 1 M HCl. After the solvent was removed, the residue was redissolved in acetonitrile:water; 1:1, and the product was purified by reverse-phase HPLC on a C-18, 10 × 150 mm, Econosphere column (Alltech/Applied Science, Deerfield, IL). The mobile phase was a linear gradient of 0-30% solvent B in solvent A (solvent A: 0.1% trifluoroacetic acid in H2O; solvent B: 0.1% trifluoroacetic acid, 99% CH3CN, 0.9% H2O). With a flow rate of 1 ml/min, the farnesylated prelamin A peptide eluted at 30 min. Synthesis of the expected product was confirmed by electrospray mass spectrometry (molecular mass = 2227 Da). Demethylation was by base hydrolysis in methanol:water as described previously (23), and purification of the demethylated peptide was by reverse-phase HPLC on the same system used for the methylated peptide (retention time = 27 min).
The S-all-trans-geranylgeranylated peptide was synthesized (22) by reacting 180 nmol of all-trans-geranylgeranyl bromide (American Radiochemical, St. Louis, MO) dissolved in 50 µl of dimethylformamide with 190 nmol of prelamin A peptide dissolved in 350 µl of NaI-saturated triethylamine:dimethylformamide; 1:150. The reaction was for 15 min at room temperature in the dark and stopped by the addition of 16 µl of 1 M HCl. The geranylgeranylated prelamin A peptide was purified on reverse-phase HPLC as for the farnesylated peptide; retention time = 35 min. Electrospray mass spectrometry of the product gave the expected molecular mass of 2296 Da.
N-Acetyl farnesyl methyl cysteine was prepared by acid catalyzed methylation of commercial N-acetyl farnesyl cysteine (Calbiochem) as described previously (24). Purification was on normal phase HPLC (250 × 4.6 silica gel column) with a mobile phase of hexane/isopropanol (85:15) and a flow rate of 1.5 ml/min (retention time = 4.5 min).
Radioiodinated PolypeptidesRadioiodination of substrate peptides was by the IODO-GEN (Pierce) method as described by Fraker and Speck (25). Briefly, a 1 mg/ml solution of peptide was prepared in a borate buffer: pH 8.2, 6.25 mM borate, 145 mM NaCl, 0.1 mM EDTA. One hundred microliters of this peptide solution was mixed with 300 µCi of Na125I (Amersham Corp.) and incubated in IODO-GEN-lined tubes on ice for 30 min. The reaction was stopped by the addition of 2 µl of 1 M dithiothreitol. The product was separated from unreacted iodine by elution from a P2 desalting column with 10 mM MES (pH = 6.0) buffer containing 2 mM KI and 0.2 mM EDTA. Typical specific activity of the isolated iodinated peptide was around 0.5 mCi/µmol.
Endoprotease AssayNuclei were prepared from HeLa cells as a source of enzyme activity. Cells were harvested by trypsinization and then washed two times with ice-cold phosphate-buffered saline. All subsequent steps were carried out at 4 °C. The cell pellet was resuspended to a final density of 4 × 108 cells/ml in a lysis buffer (0.01 M Tris-HCl, pH 7.0, 0.01 M NaCl, 3 mM MgCl2, 0.4% Nonidet P-40) reported to leave nuclei intact (26). Nuclei were isolated after two more washes, in the same buffer, and pelleting by centrifugation in a Sorvall HB-4 rotor for 10 min at 365 × g. The nuclei were resuspended in the same buffer without Nonidet P-40. Protein concentration was obtained by means of the Micro BCA protein assay reagent kit (Pierce).
The endoprotease reaction was initiated by the addition of
125I-labeled peptide (Vmax at 5 µM for farnesylated peptide) to the nuclear preparation
in a final volume of 150 µl in 10 mM MES, pH = 6.0. The reaction was run for various periods of time (linear to 90 min) at
37 °C. The reaction was stopped by the addition of 10 µl of
glacial acetic acid and chilling on ice for 10 min. The reaction mix
was then cleared by centrifugation at 2,000 rpm for 10 min in an
Eppendorf centrifuge. The supernatant was collected and lyophilized,
the residue resuspended in 25 µl of water and applied to reverse
phase thin layer chromatography plates (Analtech, Inc. Newark, DE). TLC
plates were developed in 10% acetonitrile in water, and the spots were
visualized by autoradiography. A synthetic, iodinated RSY peptide
standard was run on each plate to aid in the identification of the
expected product. The amount of labeled RSY formed in the assay was
determined by scraping the appropriate spots into tubes and
quantitation of radioactivity with a counter.
Wild-type and nonfarnesylatable mutant (MSIM)
prelamin A cDNAs cloned into the
EcoRI(5)-BamHI(3
) of the SV-40 based expression vector, pECE, were kind gifts of Dr. F. McKeon (Harvard Medical School)
and have been previously described (10, 27). The prelamin A mutant
terminating in the CAAX sequence CVLL was a kind gift of Dr.
Paul Kirschmeier (Schering-Plough Research Institute). This mutant was
prepared by means of the pAltered sites mutagnesis kit (Promega,
Madison. WI). Wild-type prelamin A cDNA was cloned into pAlter
between the EcoRI and XbaI sites. The mutagnesis
protocol was that described by Kramer et al. (28) as
modified by Promega and carried out according to the manufacturer.
Sequence verification in the mutant was by the Sequenase (U. S.
Biochemical Corp., Cleveland, OH) dideoxy sequencing method. The mutant
cDNA was subcloned into the cytomegalovirus promotor-based
expression vector pcDNA3 (InVitrogen, San Diego, CA) between the
EcoRI and XbaI sites within the polylinker. Transient transfections were by the Lipofectin method (Life
Technologies, Inc.) as described previously (10).
As described above, experiments in other laboratories (16, 17) indicate that human prelamin A is endoproteolytically cleaved between Tyr657 and Leu658. In an effort to determine what other features of the prelamin A primary sequence might play a role in substrate determination, we compared the prelamin A sequences reported for several vertebrate species. The results of such a comparison (Table I) suggest that at least three amino acid residues on either side of the cleavage site, the amino acid sequence RSYLLG, may be conserved across species lines. A search on the Swiss-Prot data base (release 33) for the sequence RSYLLG did not reveal this sequence in any protein except prelamin A. This observation reinforced the proposition that this sequence is important for recognition by the prelamin A endoprotease.
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Our prior studies (11) identifying intermediates in the prelamin A processing pathway (Fig. 1), also suggested that the endoproteolysis substrate possessed a farnesylated and methylated cysteine at the carboxyl terminus. We thus predicted that peptide I (Structure 1) would be a suitable substrate for the human prelamin A endoprotease where Y* is a radioiodinated tyrosine. If this were an appropriate substrate for the prelamin A endoprotease, the tripeptide RSY* would be released. Synthesis of the methylated apopeptide was achieved by solid state methods followed by carboxyl-terminal methylation with 5% HCl, MeOH as described under "Experimental Procedures." After HPLC purification of the apopeptide, the cysteine was farnesylated by reaction with farnesyl bromide under mildly basic conditions and repurified by reverse phase HPLC. Synthesis of the farnesylated peptide was confirmed by electrospray mass spectrometry (data not shown), and the peptide was then radioiodinated by the IODO-GEN method.
[View Larger Version of this Image (6K GIF file)]Structure 1.
The radioiodination was expected to permit quantitative monitoring of
product formation for in vitro enzyme assay. Utilizing crude
nuclear extracts from HeLa cells as a source of enzyme, formation of
the expected RSY* product was detected by reverse phase thin
layer chromatography. No product was formed when heat treated nuclear
extracts were used (Fig. 2). The co-migration of the
proteolytic product with synthetic radioiodinated RSY on thin layer was
also confirmed by HPLC. The putative RSY* was eluted from
the TLC plate and mixed with unlabeled RSY. These samples were
subjected to reverse phase HPLC and co-migration of the radiolabeled
proteolysis product and the mass standard was demonstrated (Fig.
3). These observations indicated that an assay for the
prelamin A endoprotease utilizing I as a substrate was
feasible. Release of RSY* was time dependent (Fig.
4). We examined the temperature (optimum = 37 °C) and pH dependence (optimum = 6) of the reaction as well,
leading to the standard assay conditions described under
"Experimental Procedures."
Kinetic Behavior of the Prelamin A Endoprotease
We now sought
to determine whether the proteolytic release of RSY from I
was well behaved kinetically and correlated with in vivo
observations on prelamin A endoproteolysis. Examination of the
substrate dependence of the reaction (Fig.
5A) indicated that the reaction exhibited
classical Michaelis-Menten kinetics with a Km of
0.67 µM and a Vmax of 6.5 pmol/min/mg of protein. Under Vmax conditions,
with crude nuclear extract as a source of enzyme, the assay was linear
with enzyme protein from 20 to 175 µg/assay (Fig. 5B).
These results indicate that the enzyme assay for the putative prelamin
A endoprotease is well behaved and is suitable for further study of the
activity.
To examine the specificity of the protease for the isoprenoid moiety,
we synthesized the geranylgeranylated analogue of I from the
prelamin A apopeptide and geranylgeranyl bromide, as described under
"Experimental Procedures." Preliminary experiments demonstrated
that the radioiodinated, geranylgeranylated prelamin A peptide gave
rise to RSY in endoprotease assays. That these two substrates were
being proteolyzed by the same enzyme was demonstrated by determining
that the geranylgeranylated peptide is a competitive inhibitor for
cleavage of the farnesylated peptide substrate (Fig. 6).
We then examined the activity of radioiodinated geranylgeranyl prelamin A peptide as a substrate in detail. A comparison of the kinetic parameters of the farnesylated and geranylgeranylated peptides is shown in Table II. Since these assays are performed with identical amounts of the same crude enzyme preparation, the ratio Vmax/Km gives a measure of the relative efficiency with which the endoprotease can utilize the two substrates. It thus appears that the farnesylated substrate is approximately 19-fold more reactive with the prelamin A endoprotease than the geranylgeranylated substrate.
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Lower reactivity with the geranylgeranylated substrate was also
demonstrated in whole cells by transfection studies. Chinese hamster
(CHO-K1) cells were transiently transfected with CAAX box
mutant (MSIM or CVLL) or wild-type (CSIM) human prelamin A constructs.
The MSIM sequence cannot be prenylated, whereas the CVLL sequence has
been shown to produce geranylgeranylation of other proteins (29).
Geranylgeranylation of the CVLL-prelamin A was confirmed by
demonstrating efficient [3H]mevalonate labeling of the
protein (Fig. 7) in the presence of the farnesyl protein
transferase inhibitor, BZA-5B (30). We have previously reported that
BZA-5B effectively inhibits the incorporation of
[3H]mevalonate into the farnesyl substituent of wild-type
prelamin A (15).
We next compared the proteolytic conversion of the wild-type and mutant
prelamin A. As expected, there was no prelamin A detected in cells
transfected with the wild-type construct whereas, as previously
reported (10), the nonprenylated prelamin A cannot be processed to
the mature protein. In contrast to these proteins, the
geranylgeranylated mutant could be converted to mature lamin A, but
less efficiently than the wild-type protein, as indicated by the large
amount of prelamin A which accumulates in these cells (Fig.
8). The accumulation of prelamin A in cells transfected with the CVLL mutant was also confirmed by immunoprecipitation and
indirect immunofluoresence with a prelamin A-specific antibody (data
not shown).
In order to further evaluate the biological relevance of the in vitro results, the activity of the nonfarnesylated analogue of Structure 1 was examined as a substrate. Transfection studies from our laboratory (10) and others (12), with nonfarnesylatable prelamin A mutants, have demonstrated that conversion of prelamin A to mature lamin A will not occur in such mutants. Consistent with these results, our standard assay did not indicate any formation of the RSY product from the nonfarnesylated substrate(data not shown). We also examined the activity of base-demethylated I as a substrate and again observed no formation of RSY (data not shown). This finding demonstrates that in addition to farnesylation, the substrate cysteine must be methylated to be active as a substrate. This result is consistent with a prior report from our laboratory demonstrating that the maturation of prelamin A proceeds through a farnesylated and methylated cysteine intermediate (11).
Inhibitor StudiesThe studies, described above, indicate at
least two critical chemical features of the prelamin A peptide which
render it active as a substrate: the putative RSYLLG endoprotease
cleavage site and a carboxyl-terminal farnesylated methylated cysteine.
Therefore, we examined the effect of acetyl farnesyl cysteine methyl
ester (N-acetyl farnesyl methyl cysteine) (Fig.
9A) and RSYLLG (Fig. 9B) on the
in vitro formation of RSY from I. The results indicate that both compounds can inhibit formation of RSY.
N-Acetyl farnesyl methyl cysteine inhibits the prelamin A endoprotease, noncompetitively, with an apparent Ki of approximately 17 µM. We interpret this result to be consistent with a farnesyl cysteine recognition domain in the prelamin A endoprotease which is distinct from the endoproteolysis site. On the other hand, RSYLLG exhibits competitive inhibition of the prelamin A endoprotease as would be expected if this sequence binds to the active site of the enzyme. The observed Ki for RSYLLG is 0.9 µM, which is quite similar to the Km of the farnesylated prelamin A peptide.
Since RSYLLG can act as an efficient competitive inhibitor, we also examined the activity of radioiodinated RSYLLG as a substrate in the endoprotease reaction. In contrast to the nonfarnesylated prelamin A peptide, RSYLLG is, indeed, efficiently hydrolyzed by the endoprotease (Km = 0.27 µM; Vmax = 14.2 pmol/min/mg of protein).
In order to get preliminary information on the catalytic nature of the
prelamin A endoprotease we examined class inhibitors of aspartic
proteinases (pepstatin), metalloproteases (EDTA, EGTA), cysteine
proteases (leupeptin, E-64), and serine proteases (aprotinin, 3,4-dichloroisocoumarin, chymostatin, phenylmethylsulfonyl fluoride). Inhibition was only obtained with the serine protease inhibitors (Fig.
10). The negative results for the other classes of
protease inhibitors tested are not shown.
We (10, 13) and others (12) have reported that prelamin A does not
undergo processing or assembly into the nuclear lamina in the absence
of farnesylation. Farnesylated prelamin A mutated in the endoprotease
site (RSYLLG RSYRLG) has been reported by Hennekes and
Nigg (17) to localize to the nuclear periphery but not undergo
endoproteolysis to mature lamin A. Based on this finding, these workers
suggested that one function of farnesylation of prelamin A is
localization to the nuclear envelope.
The studies presented in this report are consistent with the hypothesis that another possible function for farnesylation is binding of the farnesylated and methylated prelamin A to the prelamin A endoprotease. Several observations particularly pertain to this point. The specificity of the endoprotease for farnesylation over geranylgeranylation in vitro and in whole cells is consistent with recognition of the prenyl substituent by the enzyme. Noncompetitive inhibition by N-acetyl farnesyl methyl cysteine is also consistent with a binding site on the endoprotease for the prenyl group, albeit at a site other than the active site.
The lack of cleavage of the nonfarnesylated prelamin A peptide
substrate in vitro, and the similar lack of cleavage of the prelamin A molecule in whole cells, stands in contrast to the efficient
cleavage of the hexapeptide substrate, RSYLLG. We would speculate the
basis of these observations is that the RSYLLG sequence, as a part of
the prelamin A molecule, cannot be presented to the active site of the
endoprotease, perhaps because of secondary structural constraints.
Specific binding of the farnesylated and methylated cysteine would,
thus, direct the RSYLLG sequence to the proteolytic cleavage site. That
methylation is also important for substrate reactivity is indicated by
the lack of cleavage of the demethylated prelamin A peptide. Higher
order structure in the C terminus of nonfarnesylated prelamin is
consistent with the previous finding from our laboratory that the
prelamin A peptide domain is inhibitory for prelamin A assembly into
the lamina (10). An illustration of our hypothesis for the role of
farnesylation in prelamin A endoproteolysis is shown in Fig.
11.
An important feature of this hypothesis is that we are suggesting the
existence of a farnesyl cysteinyl methyl ester binding site on the
prelamin A endoprotease. Studies of other enzymes are also consistent
with binding sites for farnesyl cysteine methyl ester. The
Ki for noncompetitive inhibition of the prelamin A
endoprotease by N-acetyl farnesyl methyl cysteine (17 µM) is essentially identical to that reported for the
apparent Ki for the noncompetitive inhibition of the
P-glycoprotein ATPase (31) by N-acetyl farnesyl methyl
cysteine. It is also comparable to to the Km values
for two other farnesylated substrates for other enzymes. These are the
"prenyl cysteine-directed -carboxymethyl transferase (32),"
which has a Km of 11.6 µM for
N-acetyl, S-farnesyl cysteine and the
"isoprenylated protein endoprotease (19, 33)," which has a
Km of 6 µM for its farnesylated oligopeptide substrate. Prenylation is required for substrate activity
with these enzymes consistent with a polyisoprenyl binding site.
Extensive structure-activity studies of inhibitors of the isoprenylated
protein endoprotease have particularly been interpreted as consistent
with a farnesyl cysteine binding site (34). However, this enzyme
differs significantly from the prelamin A endoprotease in that
nonprenylated peptides do not act as competitive inhibitors (18). It
should also be noted that the "isoprenylated protein endoprotease"
is not affected by serine protease inhibitors (34) and is, therefore,
almost certainly distinct from the enzyme described in this report.
It has been postulated (35) that protein prenylation serves as "a
mediator of protein-protein interactions" rather than acting as a
hydrophobic anchor to lipid bilayer membranes. The data presented here
for the prelamin A endoprotease, as well as the prior studies of the
S-prenylcysteine -carboxymethyl transferase and
isoprenylated protein endoprotease, are clearly supportive of this
hypothesis.
The existence of such a polyisoprenoid recognition domain in various
enzymes is also consistent with a discrimination between polyisoprenoid
substituents in biological processes. We observe in our current studies
a difference in the rate of endoproteolytic cleavage of farnesylated
and geranylgeranylated substrates both in whole cells and in
vitro. Such dependence of substrate activity on the isoprenoid
substituent has also been reported for the S-prenylcysteine -carboxymethyl transferase (22, 32) and the isoprenylated protein
endoprotease (33).
Similarly, functional specificity of farnesylation relative to geranylgeranylation has been demonstrated for mammalian p21ras in cell growth (29), yeast RAS2 activation of adenylate cyclase (36), light-regulated association of rhodopsin kinase with ROS membranes (37) and yeast a-factor induction of mating (38). It is, therefore, intriguing to speculate that a general function of the farnesyl residue is to bind to specific sites on other proteins.