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INTRODUCTION |
Several proteins have been found to be prenylated and methylated
at their carboxyl-terminal ends (1, 2). Prenylation is a
post-translational modification directed by one of three motifs. The
Rab family of Ras-like GTPases are geranylgeranylated on the cysteine
residues of CC or CXC motifs, where C is a cysteine and
X is any amino acid. The final cysteine of the
CXC motif is also carboxymethylated. Other prenylated
proteins have a CaaX motif, where C is a cysteine, a is any
aliphatic amino acid, and X is an amino acid that specifies
whether a farnesyl (in which case X is a serine, alanine,
methionine, or glutamine) or a geranylgeranyl (in which case
X is a leucine) group will be covalently attached. Following
the attachment of the isoprenoid group, the final -aaX amino
acids are cleaved, and the cysteine residue is methylated. Prenylation
was initially believed to be important only for membrane attachment, but the 15-carbon farnesyl moiety alone does not stably anchor proteins to membranes (3-5). Another role for prenylation appears to be its importance in protein-protein interactions (6).
The only nuclear proteins known to be prenylated in mammalian cells are
prelamin A and B-type lamins. Both are farnesylated and
carboxymethylated, but prelamin A is further processed by endoproteolysis to mature lamin A, which lacks the final 18 amino acids, including the modified cysteine residue (7-10). Prenylated prelamin A containing a mutation that prevents its endoproteolytic processing can still be incorporated into the nuclear lamina (11). Unprenylated prelamin A accumulates within the nucleus with some incorporation into the nuclear lamina in cells where farnesyl synthesis
has been blocked (9, 12). Once farnesyl synthesis is restored, prelamin
A is rapidly farnesylated and cleaved into its mature form. Recently,
the prelamin A endoprotease activity was characterized from HeLa cell
nuclear extracts (13). For proteolysis to occur, prelamin A must be
farnesylated and methylated, and the lack of either modification
prevents maturation to lamin A. The specific protein or proteins
involved in this endoproteolysis are not yet isolated. Furthermore, the
cellular role of the prenylated prelamin A precursor is unknown. As a
first step toward understanding its function, we searched for proteins
that interacted with prelamin A. We now report the discovery of a novel
protein that binds the farnesylated prelamin A carboxyl-terminal domain
(preAct).1 We have named this
protein the nuclear prelamin A recognition factor, or Narf.
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EXPERIMENTAL PROCEDURES |
Plasmid Construction--
preAct contains amino acids 389-664
of prelamin A, which includes the CaaX motif (14). Act C 
S has amino acid 661 changed from a cysteine to a serine residue.
Act
CaaX contains amino acids 389-660 with an artificial
stop codon inserted after the amino acid 660 codon in the corresponding
cDNA sequence. Act contains amino acids 389-646, and the
carboxyl-terminal tail domain of lamin C (Cct) contains amino acids
389-572, which includes the 6 amino acids distinct from lamin A that
arise from alternative mRNA splicing (15). The carboxyl-terminal
tail domain of lamin B1 (Bct) contains amino acids 391-586 (16).
All cDNAs corresponding to lamin tail domains were amplified by
polymerase chain reaction (PCR) using primers containing specific restriction endonuclease sites. The cDNA sequence coding for Act C
S was generated using a PCR primer where the cysteine codon TGC was
replaced by the serine codon AGC. PCR products were either digested
with the desired restriction endonucleases or subcloned into the TA
overhang vector pCR2.1 (Invitrogen) and subsequently digested.
Amplified cDNAs were purified on agarose gels and ligated into
their respective plasmids using standard methods. The cDNA sequences corresponding to the A-type lamin tail domains were cloned
into pGBT9 (CLONTECH) in-frame with the GAL4 DNA
binding domain cDNA sequence using EcoRI and
BamHI restriction sites, and the constructs were used in
yeast two-hybrid assays. These cDNAs were also cloned into pGAD424
(CLONTECH) using SmaI and BamHI restriction sites and then further subcloned after
digestion with SmaI and SalI into pBFT4 (a gift
from Dr. J. Licht, Mt. Sinai School of Medicine, New York) in frame
with the FLAG epitope tag coding sequence. Bct cDNA was cloned into
pGBT9 in frame with the GAL4 DNA binding domain cDNA sequence using
BamHI and SalI restriction sites and subcloned
into pBFT4 in frame with the FLAG epitope tag by digesting pCR2.1-Bct
with SmaI and EcoRI.
Narf cDNA was amplified by PCR from a clone in pGADGH
(CLONTECH) using a sense primer corresponding to
untranslated cDNA sequences 
63 nucleotides from the putative ATG
translational start site and a M13+ antisense primer corresponding to
sequences in pGADGH downstream of the polyadenylation signal. This PCR
product was digested with EcoRV and XhoI and
cloned into pBFT4 in frame with the FLAG epitope tag coding sequence.
FLAG-Narf cDNA was subcloned into pGEX-4T-1 (Amersham Pharmacia
Biotech) in frame with the glutathione S-transferase (GST)
cDNA sequence or into the mammalian expression vector pSVK3
(Amersham Pharmacia Biotech) by digesting pBFT4-Narf with
BamHI and XhoI or with XbaI and
XhoI, respectively. All constructs were sequenced at the
Columbia University Cancer Center DNA Sequencing Facility. All plasmid
DNA for bacterial and yeast transformations or tissue culture cell
transfections was purified using the Wizard Plus Maxiprep Kit (Promega).
Yeast Two-hybrid Assay and Narf Sequencing--
The yeast
two-hybrid screen (17) was performed according to the manufacturer's
instructions (CLONTECH Protocol Handbook no.
PT3024-1) except that yeast transformations were carried out as
described (18). Saccharomyces cerevisiae Y190 cells were co-transformed with pGBT9-preAct as bait and a HeLa cell cDNA library (CLONTECH) as "prey." Five independent
library constructs formed blue colonies and were isolated after
screening 5 × 105 yeast colonies. Each positive Narf
clone was sequenced at the Columbia University Cancer Center DNA
Sequencing Facility using 16 different primers spaced 100-200 base
pairs apart, which yielded complete and overlapping sequences of both
strands of Narf cDNA.
Sequence Analysis Computer Programs--
BLAST, GAP, and Pileup
sequence analyses were performed using the NCI-Frederick Biomedical
Supercomputing Center's facilities (Frederick, MD). All alignments
were created using the Pileup program with default settings (gap
creation penalty = 8; gap extension penalty = 2). Percentages
of identical or similar amino acids were determined by GAP aligning
protein sequences to the deduced amino acid sequence of Narf using the
settings described above. Figures were created using MacBoxshade,
version 2, downloaded from the Internet. The following amino acid
groups were considered similar: aliphatic hydrophobic, A/I/L/M/V;
aromatic hydrophobic, F/Y; basic, K/H/R; acidic, D/E; aliphatic
alcoholic, S/T; and polar amides, N/Q.
5'-Rapid Amplification of cDNA Ends (5'-RACE)
Reaction--
5'-RACE (19) reactions were performed on the longest
Narf construct according to the manufacturer's instructions (Life
Technologies, Inc.). Briefly, HeLa cell mRNA was isolated using
TRIzol Reagent (Life Technologies) as described by the manufacturer. An
antisense sequencing primer within the first 750 base pairs of Narf
cDNA was used to amplify a single strand of antisense cDNA from
this HeLa cell mRNA. After degrading the mRNA, terminal
deoxynucleotidyl transferase was used to ligate dCTP to the 3'-end of
this single-stranded cDNA. Anchoring primers recognizing the
poly(dC) stretch and a nested antisense sequencing primer were used to
PCR-amplify the cDNA, which was then cloned into pCR2.1 and
sequenced as described above using T7 and M13
primers.
Three initial antisense primers were used, and each yielded the same 5'
cDNA sequence.
Northern Blot Analysis--
Human cancer cell line and human
tissue Northern blots were purchased from CLONTECH.
Probes were made from linear cDNA fragments of Narf, lamin B1,
actin, or a shared domain of lamins A and C using the Random Priming
System Kit (New England Biolabs) as specified by the manufacturer.
Probes were labeled with [
-32P]deoxycytidine
5'-triphosphate (NEN Life Science Products; 10 mCi/ml, 3000 Ci/mmol).
Blotting and washing of membranes were performed using standard
methods, and blots were exposed to Kodak XAR x-ray film with
intensifying screens at 70 °C for varying times.
Tissue Culture and Cell Fractionation--
HeLa cells were grown
to confluence in Dulbecco's modified Eagle's medium containing 4.5 g/liter glucose and L-glutamine (Media-Tech) supplemented
with 10% fetal bovine serum, 100 units/ml penicillin, and 100 µg/ml
streptomycin (Life Technologies). Cells were harvested by trypsin
digestion and washed with phosphate-buffered saline (PBS). Pelleted
cells were resuspended in 4 pellet volumes of hypotonic lysis buffer
(10 mM Tris-HCl, pH 7.5, 1 mM
MgCl2, 1 mM dithiothreitol, 10 µM
phenylmethylsulfonyl fluoride), allowed to swell on ice for 5 min, and
sheared by 5-7 passages through a 27-gauge needle. Plasma membrane
breakage was checked using an Olympus CK tissue culture inverted
microscope. Lysed cells were underlaid with hypotonic lysis buffer
containing 30% sucrose and centrifuged at 10,000 × g
for 10 min at 4 °C. The supernatant was removed and clarified from
nuclear or whole cell debris by centrifugation for 10 min at
14,000 × g in a microcentrifuge. This cytoplasmic
fraction was removed from the pellet and set aside. The sucrose pellet
containing nuclei was resuspended in 1 pellet volume of nuclear
extraction buffer (20 mM Tris-HCl, pH 7.5, 0.5 mM MgCl2, 1 mM dithiothreitol, 10 µM phenylmethylsulfonyl fluoride) and digested with 1 µg/ml DNase I and 10 µg/ml RNase A at room temperature for 15 min.
Nuclei in all subsequent steps were pelleted by centrifugation at
14,000 × g for 20 s. Nuclear envelopes were
washed three times with 1 pellet volume of nuclear extraction buffer
containing 0.5 M NaCl, and supernatants were saved and
combined. Nuclear envelope pellets were then washed and resuspended in
1 pellet volume of nuclear extraction buffer and sonicated briefly with
a Sonic Dismembrator (Fisher) to suspend the nuclear envelopes.
Protein concentrations of each cellular fraction were estimated by dot
blotting samples onto nitrocellulose strips and staining with Ponceau S
(Sigma). Each fraction was diluted to approximately the same
concentration in SDS sample buffer (0.125 M Tris-HCl, pH
6.8, 20% glycerol, 2.1% SDS, 0.0025% bromphenol blue, 5%
-mercaptoethanol) and stored at 20 °C. Proteins in fractions
were separated by SDS-polyacrylamide gel electrophoresis with a Bio-Rad
Mini Trans-blot Cell. Gels were Coomassie Blue-stained and destained
using standard methods and dried onto Bio-Design Gel-Wrap using a
Bio-Rad model 583 Gel Dryer.
Preparation of GST Fusion Proteins--
Escherichia
coli strain DH5 bacteria were transformed with pGEX-4T-1 or
pGEX-Narf and grown overnight. Overnight cultures were diluted 1:10
into fresh Luria broth and grown for 2-4 h at 37 °C. Protein
expression was induced by adding
isopropyl--D-thiogalactoside (Fisher) to a final
concentration of 0.5-1.0 mM and growing the bacteria for
an additional 6-18 h. Bacterial lysates were prepared, and GST or
GST-Narf fusion proteins were batch-purified using glutathione-Sepharose 4B (Amersham Pharmacia Biotech) as described in
the manufacturer's instructions. For Western blot analysis, fusion
proteins were eluted from the glutathione-Sepharose 4B beads with 10 mM reduced glutathione, 50 mM Tris-HCl, pH 8.0, and stored at 20 °C. For binding assays, GST or GST-Narf remained attached to the beads and was stored at 4 °C and used within 24 h.
Preparation of anti-Narf Antibodies--
GST-Narf fusion protein
was purified as described above and lyophilized for injection into two
rabbits (Pocono Rabbit Farm & Laboratory). Rabbits were boosted using
approximately 150 µg of GST-Narf in SDS-polyacrylamide gel slices.
Anti-Narf polyclonal antibodies were affinity-purified as described
(20) except that nitrocellulose strips were processed as described
below and antibodies were eluted with 0.2 M glycine, pH
2.8, 1 mM EGTA for 20 s.
Western Blotting--
Proteins were separated on 10%
SDS-polyacrylamide minigels and blotted onto Nitrobind nitrocellulose
membranes (Micron Separations) using a Bio-Rad Trans-blot SD Semi-dry
Transfer Cell. Efficiency of transfer was checked by staining Western
blots with Ponceau S. Blots were incubated in blocking solution (PBS,
0.1% Tween 20, 5% dried nonfat milk) for 1 h at room temperature
or overnight at 4 °C. Antibodies were diluted into fresh blocking
solution and incubated with the blots for 1 h at room temperature.
Rabbit sera with polyclonal antibodies against lamins A and C or lamin B1 (21) were diluted 1:500 and 1:1000, respectively. Preimmune and
anti-Narf rabbit sera were diluted 1:500. Affinity-purified anti-Narf
antibodies were diluted 1:20. Blots were washed for 2 × 10 min
with blocking solution and for 10 min with PBS-T (PBS, 0.1% Tween 20)
and subsequently incubated for 45-60 min with horseradish peroxidase-conjugated donkey anti-rabbit IgG antibodies (Amersham Pharmacia Biotech) diluted 1:5000 in PBS-T. Unbound antibody was removed by washing once with PBS-T for 15 min and then 4 × 5 min. Bound antibody was detected using ECL Western blotting detecting reagents, and blots were exposed for varying times to Hyperfilm ECL
chemiluminescent film (Amersham Pharmacia Biotech).
Liquid -Galactosidase Assay--
S. cerevisiae
Y187 cells were used for yeast two-hybrid liquid -galactosidase
assays according to the manufacturer's protocol (CLONTECH Protocol Handbook PT3024-1). Yeast
colonies were co-transformed with pGBT9 constructs containing cDNAs
for preAct, Act C S, ActCaaX, Act, Bct, Cct, and the
pGADGH-Narf construct described above. Yeast cell homogenates were
incubated with
o-nitrophenyl--D-galactopyranoside (Sigma)
overnight. The reaction was then stopped, and the optical density
(
= 600) was measured on a Beckman DU-6 spectrophotometer. Cultures from six yeast colonies were assayed for each data point, and
statistical significance was calculated using a paired two-sample of
the means t test.
In Vitro Binding and Prenylation Assays--
All lamin tail
domain cDNAs were transcribed into mRNA using the RiboMAX T7
Large Scale Production System (Promega) following the manufacturer's
instructions. Both [3H]mevalonolactone- and
[35S]methionine-labeled proteins were synthesized using
the Promega Nuclease Treated Rabbit Reticulocyte Lysate System. For
proteins labeled with [35S]methionine, approximately 2 µg of mRNA was translated in the presence of 40 µCi of
L-[35S]methionine (NEN Life Science Products;
10 mCi/ml, 1175 Ci/mmol) and varying amounts of FPT-II (Calbiochem) in
a 50-µl reaction volume at 30 °C for 90 min. Immediately following
translation, 5 µl of the reaction suspensions were put into SDS
sample buffer and stored at 20 °C. Two 20-µl aliquots of each
reaction were incubated with 50 µl of either GST (approximately 10 µg) or GST-Narf (5-10 µg) fusion protein coupled to
glutathione-Sepharose 4B beads as described above. Binding assay
samples were brought up to a total volume of 300 µl with PBS and
rotated at 4 °C for 90 min. Beads were pelleted and washed three
times with 300 µl of PBS. Bound proteins were eluted with SDS sample
buffer, stored at 20 °C, and electrophoresed on 10 or 12.5%
SDS-polyacrylamide gels. Gels were fixed in 5% methanol, 7.5% acetic
acid, dried, and exposed to Kodak XAR x-ray film overnight at
70 °C.
For proteins labeled with [3H]mevalonolactone, each
25-µl translation reaction contained 1 µg of mRNA, 35 µCi of
RS-[5-3H]mevalonolactone (NEN Life Science Products; 1 mCi/ml, 24 Ci/mmol) and varying amounts of the farnesyltransferase
inhibitor FPT-II. The ethanol solvent for
RS-[5-3H]mevalonolactone was removed using a Speed-vac
(Savant Instruments) at room temperature, and the dried
[3H]mevalonolactone was immediately resuspended in the
in vitro translation reaction mix. Translation took place at
30 °C for 90 min and was stopped by the addition of an equal volume
of SDS sample buffer. All translated proteins were electrophoresed on 10 or 12.5% SDS-polyacrylamide gels. Gels were fixed with isopropyl alcohol/water/acetic acid (25:65:10) for 30 min, impregnated with the
Amplify fluorographic reagent (Amersham Pharmacia Biotech) for 20 min,
dried, and exposed to Hyperfilm ECL chemiluminescent film overnight at
70 °C.
For comparison of binding affinities, autoradiographs were scanned with
an Epson Expression 636 scanner, and mean densities of each band were
calculated with NIH Image, version 1.61, which was downloaded from the
National Institutes of Health on the World Wide Web. Total reaction
values were calculated by multiplying the mean density of each
translation reaction band by 4. This value reflects the total amount of
labeled protein added to each binding assay tube. Bound protein values
measure the amounts of labeled protein that specifically associated
with GST-Narf and were calculated by subtracting the mean density of
GST-bound bands from the mean density of GST-Narf-bound bands. Ratios
of bound/total reaction were calculated by dividing the bound protein
values by the total reaction values for each sample.
Tissue Culture and Cell Transfection--
HeLa cells were
transfected using the Electroporator II (Invitrogen) following the
manufacturer's protocol. Briefly, cells were grown to approximately
80% confluence, harvested by trypsin digestion, and resuspended to a
density of 12-16 × 106 cells/ml in Dulbecco's
modified Eagle's medium without fetal bovine serum or
L-glutamine (Life Technologies). Each transfection reaction
contained 250 µl of cells and 20 µg of pSVK3-FLAG-Narf in a 0.1-cm
electroporation cuvette. Cells were incubated on ice for 10 min,
electrically shocked, and incubated for 10 min at room temperature.
Transfected cells were diluted into fresh Dulbecco's modified Eagle's
medium with L-glutamine, 10% fetal bovine serum, and
penicillin/streptomycin and grown on Nunc Lab-Tek two-chamber slides
for 24 h. Tissue culture medium was changed, and cells were grown
for an additional 48 h prior to fixation and immunofluorescence staining.
Immunofluorescence Microscopy--
Transfected HeLa cells were
washed three times with PBS, fixed with methanol at 20 °C for 6 min, and permeablized with PBS containing 0.5% Triton X-100 for 2 min
at room temperature. Fixed cells were washed for 3 × 3 min with
solution A (PBS-T, 2% normal goat serum (Sigma)). Primary antibodies
were diluted into solution B (PBS-T, 10% normal goat serum), and cells
were incubated with the antibodies at room temperature for 1 h.
Transfected cells expressing the FLAG-tagged Narf were detected using
the mouse M5 anti-FLAG monoclonal antibody (Sigma) diluted 1:200.
Rabbit anti-lamins A and C or anti-lamin B1 sera were diluted 1:100
and 1:200, respectively. Following incubation with the primary
antibodies, cells were washed 4 × 3 min with solution A. The
secondary antibodies were lissamine rhodamine-conjugated goat
anti-mouse IgG and fluorescein-conjugated goat anti-rabbit IgG (Jackson
Immunoresearch Laboratories), and both were diluted 1:100 in solution
B. Cells were incubated with the antibodies at room temperature for
1 h and then washed for 4 × 3 min with solution A and 3 × 10 min with PBS and allowed to air-dry. Coverslips were mounted over
the adherent cells using the Slowfade Light Antifade Kit, Component A
(Molecular Probes, Inc.) and sealed with clear nail polish. Slides were
examined immediately or stored at 20 °C. Immunofluorescence
microscopy was performed on a Zeiss CSM410 confocal laser scanning
system attached to a Zeiss Axiovert 100TV inverted microscope. Images were processed using Adobe Photoshop 4.0 software on a Macintosh G3 computer.
Other Chemicals--
Unless otherwise indicated, routine
chemicals were obtained either from Fisher or Sigma. Enzymes for DNA
cloning were obtained from New England Biolabs.
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RESULTS |
Isolation of a Novel preAct Binding Protein--
We screened a
HeLa cell cDNA library using preAct as the bait in a yeast
two-hybrid assay. Of approximately 5 × 105 colonies
screened, five grew on selective medium and showed -galactosidase activity on a colony filter lift assay. When the "prey" library plasmids from these colonies were isolated and sequenced, three were
found to encode the same protein, and the corresponding cDNAs differed slightly from each other only in 5'- and 3'-untranslated regions. The longest cDNA clone was 1560 base pairs and was
completely sequenced in both directions. Using a 5'-RACE reaction, we
obtained an additional 28 base pairs of upstream sequence, yielding a
complete cDNA of 1588 base pairs (Fig.
1). Comparison of this clone with the
human EST data base did not find any ESTs with additional upstream
sequences, further suggesting that we had the full-length cDNA
sequence. We named the gene product of this cDNA clone Narf, for
nuclear prelamin A recognition
factor. The assigned initiation codon appears to be the
actual start codon, yielding a 456-amino acid protein with an expected
molecular mass of 51,156 Da and a theoretical pI of 6.63. The stop
codon occurs at base pair 1369 and is followed approximately 90 base
pairs downstream by a polyadenylation signal, which is located 26 base
pairs before the polyadenylate tail. When the cDNA is translated
into a protein sequence, Kyte-Doolittle hydropathy analysis showed that
Narf is not an integral membrane protein (data not shown).
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Fig. 1.
The cDNA and deduced amino acid sequences
of Narf. The cDNA sequence includes the 1560 nucleotides
isolated from a HeLa cell cDNA library clone and the additional 28 nucleotides upstream of the coding region derived from a 5'-RACE
reaction. The first translated ATG, TGA stop codon, and polyadenylation
signal are shown in bold letters. Amino acids 13-16 or
360-363 are potential nuclear localization signals and are
underlined. This sequence has been deposited in
GenBankTM under accession number AF128406.
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Narf is Similar to Bacterial Iron Hydrogenases and Eukaryotic
Proteins of Unknown Function--
The deduced amino acid sequence of
Narf was analyzed using a BLAST search. While no known proteins from
higher eukaryotes were found to be Narf orthologs, bacterial iron-only
hydrogenases and translated open reading frames (ORFs) from eukaryotic
genomic DNA sequences showed some identity. The three bacterial
hydrogenases with the most overall similarity to Narf are shown in Fig.
2A. Over the entire length of
the protein, the D chain from the Desulfovibrio fructosovorans NADP-reducing hydrogenase (HydD in Fig.
2A) is 29% identical and 38% similar. Hydrogenase-1 from
Clostridium acetobutylicum (HydA in Fig.
2A) is 30% identical and 38% similar to Narf, while the
periplasmic iron hydrogenase-1 from Clostridium pasteurianum
(Phf1 in Fig. 2A) is 31% identical and 39%
similar. Recently, the latter hydrogenase was crystallized, and its
structure was analyzed (22). Iron-only hydrogenases catalyze the
formation of H2 by combining protons and electrons (23).
Electrons are imported into the H-cluster active site through four
iron-sulfur (Fe-S) clusters. Two of these Fe-S clusters, the FS2 domain
(indicated with 
2 in Fig. 2A) and the FS4C
domain (indicated with c) do not exist in Narf, which is
a smaller protein and lacks 136 amino-terminal acids compared with the
hydrogenases. The other Fe-S clusters are the FS4A and FS4B domains
(indicated with a or b). Three of the
cysteine residues from FS4A and one cysteine from FS4B are conserved in
Narf, but they do not make up a functional Fe-S cluster. The only
functional domain conserved between Narf and the iron-only hydrogenases
are the cysteines forming the H-cluster active site (indicated by
arrows).
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Fig. 2.
Alignment of Narf with bacterial iron-only
hydrogenases and eukaryotic proteins. White letters on a black background
represent identical amino acids, and black letters on a gray background indicate
similar amino acids at the same position in three or more of the
aligned sequences. Dots show gaps in the protein alignments
that give better overall similarity. A, bacterial iron-only
hydrogenases with the most overall homology to Narf from a BLAST
sequence comparison. From top to bottom,
sequences are as follows: Narf; D. fructosovorans
NADP-reducing hydrogenase D chain, GenBankTM accession
number D57150 (HydD; 29% identical, 38% similar to Narf);
C. acetobutylicum hydrogenase-1, GenBankTM
accession number U09760 (HydA; 30% identical, 38% similar
to Narf); C. pasteurianum periplasmic iron hydrogenase-1,
GenBankTM accession number P29166 (Phf1; 31%
identical, 39% similar to Narf); and Narf. Cysteines important for the
iron-only hydrogenase active site, or H-cluster, are indicated with
arrows. The asterisks mark conserved residues
important for the environment around the H-cluster. Other iron-sulfur
clusters are marked with carets. B, putative eukaryotic
protein sequences found to be similar to Narf using BLAST sequence
comparisons with translated genomic DNA. From top to
bottom, sequences are as follows: Narf; the translation of
Saccharomyces cerevisiae putative ORF YNL240C,
GenBankTM accession number P23503, which we have named NAR1
on the Stanford Saccharomcyes Genomic Database (NAR1; 31%
identical, 41% similar to Narf); the translation of a S. pombe ORF from cosmid C1450 (spNarf; 32% identical,
45% similar to Narf); and the translation of a C. elegans
ORF from cosmid Y54H5, Contig111 (ceNarf; 34% identical,
44% similar to Narf). Residues conserved between the putative
eukaryotic proteins, bacterial iron-only hydrogenases, and Narf are
marked with symbols corresponding to those described for
A.
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Other putative proteins with overall similarity to Narf were found by
BLAST sequence comparison of Narf with translated genomic DNA sequences
from various eukaryotes. None of the ORF translations shown has a known
function. The S. cerevisiae protein from the putative ORF
YNL240C, which we have named NAR1 on the Stanford Saccharomcyes Genomic Database (Nar1 in Fig.
2B), is 31% identical and 41% similar to Narf. A
translated ORF from chromosome 3 of Schizosaccharomyces
pombe (spNarf in Fig. 2B) is 32% identical and 45% similar to Narf, and the translation from a
Caenorhabditis elegans ORF on chromosome 3 (ceNarf in Fig. 2B) is 34% identical and 44%
similar to Narf. These putative proteins show homology to Narf in
regions different from those Narf shares with the iron-only hydrogenases; however, all four of the H-cluster cysteine residues are
conserved in all of the proteins. Narf has approximately the same
percentages of identical and similar amino acids with the eukaryotic
and bacterial proteins, but the eukaryotic proteins are more similar in size.
Characterization of Narf mRNA Expression and Protein
Subcellular Localization--
The mRNA transcript size and
expression pattern of Narf was determined by probing human cancer cell
line and human tissue Northern blots with 32P-labeled Narf
cDNA (Fig. 3). Only one Narf mRNA
transcript of 2 kilobases was detectable on both Northern blots, which
is large enough to encode a peptide of the size predicted by the
cDNA clone isolated from the yeast two-hybrid assay. Various
mRNA expression patterns in several human tissue culture cell lines
are shown in Fig. 3A. All cell lines show Narf expression,
although mRNA levels vary. The colorectal adenocarcinoma SW480 cell
line showed the highest Narf mRNA expression, while lung carcinoma
A549 cells and melanoma G361 cells had the lowest levels of expression.
As expected, lamin B1 is expressed in all cell types, while lamins A
and C are variably expressed. No lamin A/C transcripts are detected in
Burkitt's lymphoma Raji cells, while barely detectable levels are seen
in promyelocyte leukemia HL-60 cells and lymphoblastic leukemia MOLT-4
cells. Narf expression is unrelated to lamin A/C expression, since all
three of these cell lines show high levels of Narf mRNA. Similarly,
high levels of lamin A/C expression in human tissues such as the
placenta (Fig. 3B) do not correlate with high Narf
expression levels. Narf mRNA is predominantly expressed in skeletal
muscle, heart muscle, and brain, although all tissues show some Narf
transcripts.
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Fig. 3.
Northern blot analysis of Narf, lamins A and
C, lamin B1, and actin mRNA expression in human cancer cell lines
and tissues. From top to bottom, estimated
mRNA sizes are as follows: Narf, 2 kilobases; lamin A, 2.9 kilobases; lamin C, 2 kilobases; lamin B1, 3 kilobases; and actin, 2 kilobases with an additional 1.8-kilobase isoform seen in skeletal and
heart muscle tissue samples. Actin is included as a control showing
mRNA loading for each lane. A, from left to
right, human cancer cell lines represented on the Northern blot
are as follows: promyelocytic leukemia HL-60, HeLa cell S3, chronic
myelogenous leukemia K-562, lymphoblastic leukemia MOLT-4, Burkitt's
lymphoma Raji, colorectal adenocarcinoma SW480, lung carcinoma A549,
and melanoma G361 cells. B, from left to
right, human tissue samples on the Northern blot are as
follows: heart, brain, placenta, lung, liver, skeletal muscle, kidney,
and pancreas.
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Anti-Narf antibodies were made in rabbits against a bacterially
expressed GST-Narf fusion protein. GST (Fig.
4A, arrowhead) and
GST-Narf (Fig. 4A, arrow) were
electrophoretically separated on an SDS-polyacrylamide gel and
Coomassie Blue-stained. When used to probe a Western blot, preimmune
rabbit serum did not detect either GST or GST-Narf (Fig. 4B,
left blot), while sera from rabbits immunized
against GST-Narf (Fig. 4B, right blot)
detected GST (arrowhead), GST-Narf (arrow), and
degradation products. To determine whether Narf was expressed in
vivo, HeLa cells were fractionated into cytoplasmic and nuclear
envelope fractions. The nuclear envelopes were then washed with 0.5 M NaCl to dissociate peripheral membrane proteins. Proteins
in the cytoplasmic fraction (CF), salt-washed nuclear envelopes (NS),
and salt-wash supernatants (SW) were separated on SDS-polyacrylamide
gels. Proteins from these HeLa cell fractions are shown on a Coomassie
Blue-stained gel in Fig. 4C. Anti-Narf antibodies were
affinity-purified against the bacterially expressed GST-Narf antigen.
When used to probe a Western blot of HeLa cell fractions, these
antibodies specifically detected a 52-kDa protein in the SW fraction
(Fig. 4D, left blot), which matches
the molecular mass predicted by translation of the Narf cDNA.
Polyclonal rabbit sera were used to probe for lamin B1 and lamin A/C
protein expression. As expected, lamin B1 was exclusively found in the
NS fraction (Fig. 4D, middle blot).
Lamins A and C were predominantly in the NS fraction, but some protein
was also found in the SW fraction (Fig. 4D, right
blot).
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Fig. 4.
Western blot analysis of bacterially
expressed GST or GST-Narf and of HeLa cell cytoplasmic and nuclear
fractions. GST or GST-Narf were expressed in bacteria, purified,
and electrophoresed on 10% SDS-polyacrylamide gels. A,
Coomassie Blue staining of GST (26 kDa, arrowhead) and
GST-Narf (86 kDa, arrow). B, Western blots were
probed with preimmune rabbit serum (left) or anti-Narf serum
(right). Preimmune serum did not detect any bacterial
proteins, while GST (arrowhead), GST-Narf
(arrow), and GST-Narf degradation products were recognized
by anti-Narf antibodies. HeLa cells were fractionated as described
under "Experimental Procedures." Approximately 10-20 µg of each
fraction was electrophoresed on 10% SDS-polyacrylamide gels. From
left to right, cellular fractions are CF, NS, and
SW. C, Coomassie Blue staining of each fraction.
D, Western blots probed with affinity-purified anti-Narf
rabbit serum (left), anti-lamin B1 rabbit serum
(middle), or anti-lamin A/C serum (right).
Anti-Narf antibodies detected a 52-kDa protein in the SW fraction that
matches the molecular mass predicted by the deduced amino acid
sequence. Narf is not cytoplasmic and can be extracted from nuclear
envelopes into the SW fraction, indicating that it is not a
transmembrane protein. Anti-lamin B1 antibodies detected a single
66-kDa protein in the NS fraction, and antibodies anti-lamin A/C
detected lamin A (72 kDa) and lamin C (65 kDa) in the NS, although some
lamins A and C were also found in the SW fraction. The migrations of
molecular mass standards in kDa are indicated at the left of
selected panels.
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Both the mRNA and protein sizes found for Narf match those expected
from its cDNA sequence. Narf does not appear to be a transmembrane protein, since it can be dissociated from the nuclear envelope by salt
treatment, which is consistent with its predicted hydrophobicity plot
data. These data show that Narf is in the nuclear fraction of cells and
is therefore in the appropriate subcellular location for prelamin A
association. Narf also appears to be expressed in cells without lamin
A. In agreement with the Northern blot data (Fig. 3), Western blot
analysis of subcellular fractions from Raji cells, which express Narf
but not lamin A/C mRNA, showed that Narf was also found in the SW
fraction of Raji cells, while no lamin A/C was detected (data not shown).
Binding Assays Confirm That Narf Specifically Binds to
preAct--
We tested the interactions of Narf with various lamin tail
domains using a yeast two-hybrid liquid -galactosidase assay (Table I). A comparison of -galactosidase
activities gives an indication of the relative binding strengths of the
bait and "prey" fusion proteins. Yeast cells were transformed with
the original Narf library clone, pGADGH-Narf, which expresses the GAL4
activation domain fused to Narf. When co-transformed with the empty
GAL4 DNA binding vector, pGBT9, yeast cells showed a background level of -galactosidase activity. Background -galactosidase activities were also measured when cells were co-transformed with Bct and Cct bait
constructs. The only significant -galactosidase activity was found
in cells co-transformed with the original preAct bait construct.
Mutation of the CaaX motif cysteine to a serine residue (Act
C S) or deletion of the CaaX motif
(ActCaaX), decreased -galactosidase activity to
background levels. Similarly, Narf apparently did not associate with
the mature Act, which lacks the final carboxyl-terminal 18 amino acids.
As S. cerevisiae cells prenylate, carboxymethylate, and
endoproteolytically remove -aaX sequences from proteins
containing CaaX motifs (2), the fact that Narf does not
associate with Act C S or ActCaaX in yeast suggests
that Narf requires that preAct be prenylated in order to bind. Narf
does not appear to indiscriminately associate with prenylated proteins,
since it does not bind Bct, which also has a carboxyl-terminal
CaaX motif.
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Table I
Lamin tail domain interactions with Narf in a yeast two-hybrid liquid
-galactosidase assay
Yeast were transformed with pGADGH-Narf and the indicated bait
constructs. One -galactosidase unit is defined as the amount of
enzyme activity required to hydrolyze 1 µmol of
o-nitrophenyl--D-galactopyranoside per min
per cell. Cultures from six different colonies were assayed for each
construct. -Galactosidase units are shown ± S.E.
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To confirm the results obtained in the two-hybrid assays, we performed
GST binding assays. Lamin tail domains cDNAs were transcribed into
mRNA using the Promega RiboMAX T7 kit, and the mRNA was
in vitro translated in the presence of
[35S]methionine using Promega nuclease-treated rabbit
reticulocyte lysates. The 35S-labeled proteins were then
incubated with GST or a GST-Narf fusion protein bound to
glutathione-Sepharose 4B beads. Beads were washed, and the proteins
eluted with SDS-polyacrylamide gel electrophoresis sample buffer were
electrophoresed on SDS-polyacrylamide gels and analyzed by
autoradiography. preAct, Act C S, ActCaaX, Act, Cct,
and Bct were efficiently translated, and roughly equal amounts of each
protein were used in the binding assay (Fig.
5A). While a barely detectable
amount of preAct bound nonspecifically to GST (Fig. 5B,
lane 1), much more was retained by GST-Narf (Fig. 5B, lane 7). Act C S and
ActCaaX also bound to GST-Narf (Fig. 5B,
lanes 8 and 9), but their binding was much weaker
than the association of preAct with GST-Narf. The other tail domains
showed very weak associations with GST-Narf (Fig. 5B,
lanes 10-12), which were not above the background level of
preAct and GST association.
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Fig. 5.
Binding of lamin tail domains to
GST-Narf. The indicated lamin tail domains were synthesized by
in vitro translation and labeled with
[35S]methionine. Gels were dried and exposed to x-ray
film. A, proteins from 10% of each in vitro
translation reaction were electrophoresed on a 12.5%
SDS-polyacrylamide gel. From left to right,
35S-labeled proteins correspond to the preAct (lane
1), Act C S (lane 2), ActCaaX (lane
3), Act (lane 4), Cct (lane 5), and Bct
(lane 6). B, 40% of each in vitro
translation reaction described in A was incubated with GST
or GST-Narf attached to glutathione-Sepharose 4B beads. Beads were
washed with PBS, and bound proteins were eluted into SDS sample buffer
and electrophoresed on 12.5% SDS-polyacrylamide gels. While some
preAct interacted nonspecifically with GST (lane 1), the
strongest interaction was between preAct and GST-Narf (lane
7). Act C S and ActCaaX also weakly bound to GST-Narf
(lanes 8 and 9), while Act, Cct, and Bct
(lanes 10-12) did not bind GST-Narf above background
levels. The migrations of molecular mass standards in kDa are indicated
at the left of selected panels.
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preAct Farnesylation Greatly Enhances Narf Binding--
Ras and
full-length prelamin A have been shown to be correctly farnesylated
using nuclease-treated rabbit reticulocyte lysate in vitro
translation reactions (9, 24, 25). We have confirmed that preAct is
also prenylated in vitro. Mevalonolactone rapidly converts
to mevalonate, a farnesyl precursor, when placed in an aqueous
environment. When [3H]mevalonolactone was added to
in vitro translation reaction mixtures, preAct was
3H-labeled (Fig.
6A, lanes 1 and
4), while Act C S and ActCaaX remained
unlabeled (Fig. 6A, lanes 2 and 3).
This prenylated 3H-labeled preAct behaved like the
[35S]methionine-labeled protein and specifically bound to
GST-Narf with no detectable association with GST in a binding assay
(data not shown).
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Fig. 6.
Inhibition of preAct farnesylation leads to a
decrease in GST-Narf binding. A, preAct (lane
1), Act C S (lane 2), and ActCaaX
(lane 3) were synthesized by in vitro translation
in the presence of [3H]mevalonolactone, and proteins were
electrophoresed on a 12.5% SDS-polyacrylamide gel that was treated
with a fluorographic reagent, dried, and exposed to x-ray film. preAct
was also synthesized in the presence of
[3H]mevalonolactone with varying amounts of the
farnesyltransferase inhibitor FPT-II, and translation products were
electrophoresed on a 10% SDS-polyacrylamide gel that was treated with
a fluorographic reagent, dried, and exposed to x-ray film. From
left to right, samples are as follows: preAct
(lane 4) and preAct synthesized with 1.5 µM (lane 5), 15 µM (lane
6), or 150 µM (lane 7) FPT-II. preAct was
labeled with [3H]mevalonolactone (lanes 1 and
4), while Act C S (lane 2) and
ActCaaX (lane 3) were not labeled. Increasing
amounts of FPT-II decreased the [3H]mevalonolactone
labeling of preAct (lanes 5-7). B, preAct and
ActCaaX tail domains were synthesized by in
vitro translation in the presence of [35S]methionine
and varying amounts of FPT-II. Proteins were electrophoresed on 10%
SDS-polyacrylamide gels that were dried and exposed to x-ray film. From
left to right, samples are as follows: preAct (lane
1); ActCaaX (lane 2); preAct with 1.5 µM (lane 3), 15 µM (lane
4), and 150 µM (lane 5) FPT-II added
during synthesis; and a control reaction (lane 6) where
preAct was synthesized without inhibitor but 150 µM
FPT-II was added during the binding reaction to ensure that the
inhibitor did not interfere with protein-protein interactions.
C, for each sample, 4-fold more reaction mixture than the
amount shown in B was incubated with GST (lanes
1-6) or GST-Narf (lanes 7-12) attached to
glutathione-Sepharose 4B. Beads were washed with PBS, and bound
proteins were eluted into SDS sample buffer and electrophoresed on 10%
SDS-polyacrylamide gels as described for B. FPT-II
inhibition of preAct farnesylation prevents GST-Narf binding in a
dose-dependent manner (lanes 9-11). The control
reaction where FPT-II was added during the binding assay did not affect
GST-Narf binding (compare lanes 7 and 12). The
migrations of molecular mass standards in kDa are indicated at the
left of selected panels. D, the
autoradiographs shown in B and C were analyzed by
densitometry as described under "Experimental Procedures." Ratios
of 35S-labeled proteins specifically bound to GST-Narf
confirm that preAct must be prenylated in order to bind GST-Narf. The
values shown are in arbitrary units.
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To confirm that preAct is farnesylated, increasing amounts of the
farnesyltransferase-specific inhibitor FPT-II (26) were added to preAct
in vitro translation reactions that contained [3H]mevalonolactone. Increasing FPT-II concentrations led
to a decrease in the [3H]mevalonolactone labeling of
preAct (Fig. 6A, lanes 4-7), with no detectable
labeling seen when 150 µM was added (Fig. 6A,
lane 7). The same concentrations of FPT-II were used in
preAct translation reactions containing [35S]methionine.
Twenty percent of the translation reaction used for each binding assay
was electrophoresed on an SDS-polyacrylamide gel (Fig. 6B).
Deletion of the CaaX motif or increasing amounts of FPT-II
led to a decrease in GST-Narf binding (Fig. 6C, lanes 9-11). At 150 µM FPT-II, preAct was not detectably
prenylated (Fig. 6A, lane 7), and the comparable
[35S]methionine-labeled sample was significantly impaired
in its ability to bind GST-Narf (Fig. 6C, lane
11). This decrease in binding was not due to FPT-II interfering
with protein-protein interactions, since 150 µM FPT-II
added to a preAct sample after translation but prior to the binding
reaction did not affect preAct binding to GST-Narf (Fig. 6C,
lane 12).
To compare the amounts of preAct that specifically bound to GST-Narf
upon FPT-II treatment, the autoradiographs shown in Fig. 6,
B and C, were scanned, and the bands were
analyzed by densitometry. The net ratios of tail domains attached to
GST-Narf were calculated by subtracting the background levels attached
to GST as described under "Experimental Procedures." The ratios for
each sample are shown graphically in Fig. 6D. Ratios from
three independent binding assays showed the same decrease in preAct
association with GST-Narf with increasing concentrations of FPT-II.
Nonprenylated Act C S (data not shown) and ActCaaX
clearly bind to GST-Narf much more weakly than prenylated preAct.
Similarly, in vitro translated and
[35S]methionine-labeled Narf does not bind to bacterially
expressed, and therefore unprenylated, GST-preAct (data not shown).
When Narf Is Overexpressed in HeLa Cells, It Is Nuclear and
Partially Co-localizes with the Nuclear Lamina--
Endogenous Narf
was shown to be nuclear on Western blots (see Fig. 4). To examine the
co-localization of Narf and lamin A within the nucleus, HeLa cells were
transiently transfected with a FLAG epitope-tagged Narf under the
control of the SV40 early promoter. Transfected cells were grown for a
total of 72 h, fixed, and labeled with anti-lamin A/C (Fig.
7, A and D, shown
in green) and anti-FLAG (Fig. 7, B and
E, shown in red) antibodies. FLAG-tagged Narf was
always exclusively nuclear, although fluorescence patterns varied
between individual cells. In some cells, exogenous Narf formed large
aggregates at the nuclear periphery (Fig. 7B) and co-localized with lamins A and C at the nuclear lamina
(yellow in Fig. 7C). Other cells showed a more
diffuse intranuclear staining (Fig. 7E), which also
partially overlapped with the nuclear lamina (Fig. 7F).
These fluorescence patterns probably reflect different levels of
exogenous Narf expression.
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Fig. 7.
Overexpression of FLAG epitope-tagged Narf in
HeLa cells. HeLa cells were transiently transfected with FLAG-Narf
cDNA under the control of the SV40 early promoter. After 72 h,
cells were fixed and stained with mouse anti-FLAG and rabbit anti-lamin
A/C primary antibodies and lissamine rhodamine-conjugated anti-mouse
IgG and fluorescein-conjugated anti-rabbit IgG secondary antibodies.
Immunofluorescently stained cells were analyzed using a Zeiss Axiovert
confocal microscope, and 1-µm central optical sections are shown.
A and D show lamin A/C staining in
green, B and E show FLAG-tagged Narf
expression in red, and C and F show
the overlay of both staining patterns, where yellow
indicates co-localization of the two proteins. The top right cell in A-C and the
bottom cell in D-F are untransfected
and show no anti-FLAG staining. Exogenous FLAG-tagged Narf is always
exclusively nuclear and forms aggregates near the nuclear lamina or is
distributed throughout the intranuclear space. Bars, 10 µm.
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We also attempted to determine the localization of endogenous Narf in
HeLa cells by immunofluorescence microscopy using the same
affinity-purified anti-Narf antibodies we used for Western blot
detection. Unfortunately, Narf protein levels in individual HeLa cells
are too low, or our antibodies are of too low avidity, to detect
endogenous Narf using immunofluorescence microscopy (data not shown).
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DISCUSSION |
Narf Is a Novel Nuclear Protein--
Prelamin A is farnesylated
and carboxymethylated on the cysteine residue of a carboxyl-terminal
CaaX motif (7-10). This post-translationally modified
cysteine residue is removed from prelamin A when it is endoproteolytically processed into mature lamin A, which lacks the
final 18 amino acids of prelamin A. This processing event appears to
occur within the nucleus (9, 12, 27). The prelamin A endoprotease
activity has been examined in HeLa cell nuclear extracts, and
endoproteolysis requires both farnesylation and carboxymethylation of
the prelamin A CaaX motif cysteine residue (13). The
significance of the prenylated prelamin A precursor is unclear, since
the inhibition of prelamin A processing or cellular prenylation
reactions does not prevent prelamin A from being incorporated into the
lamina (9-12). We report here a novel nuclear protein, Narf, which
binds to the prenylated prelamin A carboxyl-terminal tail domain. Narf
does not associate with the mature lamin A tail domain and only weakly
interacts with preAct mutants where the CaaX motif cysteine
has been replaced with a serine residue or where the CaaX
motif has been deleted. The inhibition of farnesyltransferase leads to
a dose-dependent reduction in preAct prenylation and a
concurrent decrease in Narf association. Unlike the prelamin A
endoprotease activity, Narf does not appear to require cysteine carboxymethylation in order to bind preAct. No significant sequence identity is seen when Narf is BLAST-aligned with other proteases, -aaX endoproteases, carboxymethyltransferases, or
farnesyltransferases from yeast or mammals. Narf may be a component of
a prelamin A endoprotease complex, and if so, it is interesting
to note that Narf mRNA and protein are expressed in cells that do
not have any lamin A.
Narf is similar to bacterial iron-only hydrogenases and putative
eukaryotic proteins, but sequence analysis of Narf does not show any
protein motifs that might indicate function. Bacterial iron-only
hydrogenases take electrons generated from pyruvate oxidation and
combine them with protons to make hydrogen gas (23). The only domain
conserved between Narf and the bacterial hydrogenases is the H-cluster
active site, a structurally unique site that coordinates six Fe-S
clusters between four cysteine residues (22). None of the other Fe-S
clusters, involved in electron transfer, are conserved between the
iron-only hydrogenases and Narf. Similarly, the cysteine residue
implicated in proton donation, cysteine 299 in C. pasteurianum hydrogenase-1, corresponds to alanine 171 in Narf.
There is no other uncoordinated cysteine residue near the conserved
H-cluster that might fill this proton donation role. We have yet to
determine whether Narf contains Fe-S clusters, but without the
components required to import electrons or supply protons, it is
unlikely that Narf is a functional hydrogenase.
None of the putative eukaryotic proteins found to be similar to Narf
have been purified or characterized. All of these proteins appear to
share the cysteine residues found in the H-cluster active site of the
bacterial iron-only hydrogenases. Four other cysteine residues are also
conserved between the eukaryotic and bacterial proteins, although they
are part of two different Fe-S clusters in the bacterial enzymes. While
the overall percentages of amino acids similar and identical to Narf
are approximately the same for the eukaryotic and bacterial proteins,
the eukaryotic proteins are more similar in length and appear to share
more clusters of amino acids with Narf than the hydrogenases. It
remains to be determined whether Narf or the eukaryotic proteins
contain Fe-S clusters or share any functions. However, the fact that
Narf has yeast orthologs such as Nar1 indicates that the protein may
have functions beyond its association with lamin A, since S. cerevisiae does not have genes encoding nuclear lamin-like proteins.
We have shown that endogenous Narf is found within the nuclear fraction
of HeLa cells and that overexpressed Narf is exclusively nuclear. The
dispersed intranuclear FLAG-tagged Narf staining pattern shows that it
is a nonmembrane protein, in agreement with predictions based upon its
amino acid sequence and the dissociation of Narf from nuclear envelopes
by 0.5 M NaCl washing. These data indicate that Narf has
all of the information necessary to be imported into the nucleus, and
possible nuclear localization signals (28) are underlined in
Fig. 1. Alternatively, Narf, at 52 kDa, is a small enough protein to
freely diffuse through the nuclear pore complex (29) and may be
retained within the nucleus by binding nuclear proteins. Lamin A
filaments have been shown to extend into the nucleus (30-32) and,
since their prenylation status is unknown, may be Narf anchors.
Farnesylation of preAct Enhances Narf Binding--
preAct
farnesylation greatly increases its association with Narf. In yeast,
where proteins are prenylated in the same manner as in mammalian cells
(2), Narf only associated with the prelamin A carboxyl-terminal tail
domain and not with the tail domain of lamin B1, which is also
prenylated. Confirmation that Narf specifically recognizes preAct, and
not just a prenylated cysteine residue, came from in vitro
binding assays. Although mutation or removal of the CaaX
motif from preAct prevented detectable binding in yeast, the high
concentrations of proteins in the in vitro binding reactions
were able to detect the weak associations of Narf with Act C S and
ActCaaX. Both yeast two-hybrid and in vitro assays, however, clearly demonstrated that prenylated preAct was greatly enhanced in its ability to bind Narf.
That preAct was specifically farnesylated and not geranylgeranylated is
suggested by data showing that rabbit reticulocyte lysates correctly
attach a farnesyl group and not a geranylgeranyl group to
p21K-ras(B) in vitro (25). Our data
showed that FPT-II, a inhibitor of farnesyltransferase, decreased the
incorporation of any [3H]mevalonolactone derivatives,
which would include farnesyl and geranylgeranyl, in a
dose-dependent manner. This inhibition of preAct
prenylation also led to a dose-dependent decrease in the ability of Narf to bind preAct. Although FPT-II treatment never brought
Narf and preAct association down to the levels seen for Narf and
ActCaaX, this result may be explained by the fact that 3H has a much lower specific activity than 35S.
While there is no detectable [3H]mevalonolactone labeling
of preAct upon 150 µM FPT-II treatment, there may be
residual prenylated proteins that are too weak to be seen on x-ray film
but are still available to bind to Narf. This small amount of protein
may have been enough to be detectable only when labeled by
[35S]methionine. The high concentration of FPT-II
required to decrease farnesylation (FPT-II has an in vivo
IC50 of 75 nM) (26) is also not surprising,
since in vitro translation reactions do not exactly
duplicate in vivo protein concentrations. Rabbit
reticulocyte lysates appear to have higher concentrations of
farnesyltransferase than that found in cells, and therefore more
inhibitor is required to prevent prenylation. A further confirmation
that Narf preferentially binds prenylated preAct occurred when the
binding assay components were reversed. In vitro translated
Narf labeled with [35S]methionine did not significantly
bind to preAct when it was synthesized as a GST fusion protein in
bacteria and, therefore, not prenylated.
The interaction of prenylated preAct with Narf does not appear to
require -aaX endoproteolysis or carboxymethylation. The rabbit reticulocyte lysates used for our in vitro
translation reactions do not contain endoplasmic reticulum and plasma
membranes, and therefore they cannot have -aaX endoprotease
or carboxymethyltransferase activities (2). Rabbit reticulocyte
lysates have been shown to correctly farnesylate, but not to
further post-translationally modify, in vitro translated
p21K-ras(B) (25). The apparent requirement
that preAct be farnesylated without a requirement for
carboxymethylation distinguishes Narf from the prelamin A
endoproteolytic activity found in HeLa cells, which required both
modifications in order to bind and cleave prelamin A (13).
Other Prenyl-dependent Binding Proteins--
The
importance of prenylation in conjunction with upstream protein
sequences for protein-protein interactions has been demonstrated for
several other proteins. Small GTPases are involved in many cellular
functions, and carboxyl-terminal prenylation is required for several of
their protein-protein interactions (33). Rab is a Ras-like GTPase
involved in intracellular membrane trafficking. The geranylgeranylation
of several Rabs is required for their association with the Rab GDP
dissociation inhibitor (GDI), a protein important for membrane
extraction and recycling of Rab proteins (34-37). Changing the prenyl
moieties attached to Rabs by substituting a farnesyl CaaX
motif in place of the normal CXC or CC geranylgeranyl motif
significantly reduces the association of prenylated Rabs with GDI (36,
38). While the final cysteine residue of the CXC motif is
normally carboxymethylated, this modification was not necessary for
geranylgeranylated Rab3A to bind its GDI (34). Residues amino-terminal
to the Rab prenylation motifs have also been found to be important for
GDI association (36), and a single amino acid point mutation in Rab1B
prevents its interaction with GDI, even when Rab1B is
geranylgeranylated (39).
Signal transduction molecules are also prenylated, and their
protein-protein interactions can be affected when the attached prenyl
moieties are changed. Farnesylated yeast Ras2 binds to and activates
adenylyl cyclase with 100 times higher affinity than unprenylated
recombinant Ras2 (40). The coupling of heterotrimeric G-proteins to
their receptors also relies upon prenylation. The 
1-subunit involved in the association of G-proteins with
activated rhodopsin must be farnesylated in order for this interaction
to occur, and geranylgeranylation could not substitute (41, 42). In
contrast, geranylgeranylated -subunits were more effective than
farnesylated -subunits in coupling the G-protein complex to A1
adenosine receptors (43).
Other protein-protein interactions require prenylation, but either
farnesylation or geranylgeranylation allows for binding. The prenylated
Rab acceptor protein-1 specifically binds prenylated Rabs, regardless
of the prenyl moiety attached (44). Mammalian Ki-Ras4B can be
geranylgeranylated or farnesylated, but it must be prenylated in order
to associate with its nucleotide exchange factor (45). Prenylated
Ki-Ras4B has also been found to bind to microtubules in
vitro, and disruption of the microtubule network in cells leads to
inappropriate Ki-Ras4B targeting (46). Actin filaments may also be
modified by proteins that must be prenylated in order to function
(47-49). These cytoskeletal prenyl-dependent interactions,
however, require that the binding protein be prenylated. Prelamin A and
Narf binding is unique in that it is the nucleoskeletal protein that
must be prenylated in order the interaction to occur.
Other Lamin A Binding Partners--
Lamins A and C are usually
expressed only in terminally differentiated cells (21, 50-57).
In vitro, lamin A can bind to DNA (58, 59), histones (60,
61), and the retinoblastoma gene product (62, 63), suggesting that
lamin A may play a role in the organization of DNA and transcription in
cells. Supporting this theory are data showing that prelamin A
accumulation within cell nuclei significantly inhibits DNA synthesis
upon lovastatin treatment (64). In contrast, overexpression of lamin A
does not increase the sensitivity of DNA synthesis to lovastatin.
Although lovastatin treatment leads to an inhibition of cellular
prenylation reactions, these data are the first to describe a direct
role for prelamin A in a nuclear function.
Narf is a nuclear protein that interacts with a prenylated partner.
Narf has no homology to other known prenyl-dependent
binding proteins, including enzymes that recognize and modify
prenylated substrates such as the -aaX endoprotease and
carboxymethyltransferase. Prenylation of preAct greatly enhances its
association with Narf, while carboxymethylation does not appear to be
required. The functional significance of the association of Narf with
prenylated prelamin A is still unknown. Since Narf is expressed in
cells that do not have any lamin A or C, it may have functions in the
cell independent of its association with preAct, and prelamin A may be
important in regulating these functions in a cell type- and
differentiation-specific manner. Alternatively, Narf may be important
in prelamin A processing, or it may anchor prenylated prelamin A in the
intranuclear space, allowing prelamin A to organize nuclear proteins
into a higher order structure. The isolation of Narf is an important
first step in determining the role of prelamin A in the cell.
TOP
ABSTRACT
INTRODUCTION
An Irma T. Hirschl Scholar. To whom correspondence should be
addressed: Dept. of Medicine, College of Physicians and Surgeons, Columbia University, 630 W. 168th St., P & S 10-508, New York, NY
10032. Tel.: 212-305-8156; Fax: 212-305-6443; E-mail:
hjw14@columbia.edu.
