1 Institute of Molecular Medicine, National Taiwan University Hospital, College
of Medicine, National Taiwan University, Taipei, Taiwan, Republic of
China
2 Institute of Pathology, National Taiwan University Hospital, College of
Medicine, National Taiwan University, Taipei, Taiwan, Republic of China
* Author for correspondence (e-mail: fangjen{at}ha.mc.ntu.edu.tw)
Accepted 23 August 2002
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: ADP-ribosylation factors, Myristoylation, Heterochromatin, Importin-, HP1
, Embryonic development
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The biological functions of ARLs are still unclear, although some are
expressed in tissue- and/or differentiation-specific patterns
(Clark et al., 1993;
Cavenagh et al., 1994
;
Schurmann et al., 1994
;
Zhang et al., 1995
;
Hong et al., 1998
). ARLl was
localized in the Golgi complex of normal rat kidney cells
(Lowe et al., 1996
) and S.
cerevisiae (Lee et al.,
1997
), consistent with a function in vesicular trafficking. Unlike
the lethal phenotype of double null alleles of arf1 and
arf2, however, knock-out of the yeast ARL1 gene was not lethal
(Lee et al., 1997
). Expression
of ARL4 was reported to be cell differentiation-dependent and developmentally
regulated (Clark et al., 1993
;
Lin et al., 2000
). ARL4, with
its distinctive nuclear/nucleolar localization and pattern of developmental
expression, was inferred to play a unique role(s) in neurogenesis and
somitogenesis during embryonic development and in the early stages of
spermatogenesis in adults (Lin et al.,
2000
).
To obtain additional clues to physiological role(s) of ARLs, we
investigated the expression, subcellular localization, and biochemical
properties of ARL5. As reported here, expression of mouse ARL5 (mARL5),
similar to that of mARL4, was developmentally regulated during embryogenesis
and was mainly detected in nuclei. When expressed in COS-7 cells, hARL5(T35N),
a mutant predicted to be GDP-bound, was localized to nucleoli. Data from yeast
two-hybrid and protein interaction analyses revealed that hARL5 interacted
with the heterochromatin protein 1 (HP1
, through its C-terminal
MIR-like motif and this interaction was nucleotide-dependent. HP1
is
one of the three mammalian HP1 family proteins that have been found in many
other organisms from Schizosacchromyces pombe
(Lorentz et al., 1994
) to
mammals (Singh et al., 1991
;
Saunders et al., 1993
).
HP1
is a nonhistone chromosomal protein suppressor of position effect
variegation in Drosophila (James
and Elgin, 1986
; Eissenberg et
al., 1990
). It is associated with heterochromatin
(James and Elgin, 1986
;
Eissenberg et al., 1990
) and
telomeres (Fanti et al.,
1998
), and prevents telomere fusion
(Fanti et al., 1998
). Thus,
ARL5, like ARL4, may have a physiological role(s) in nuclear dynamics and/or
signaling cascades during embryonic development.
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Full-length wild type hARL5 cDNA was generated using 5' (sense)
primer ARL5A, and 3' (antisense) primer ARL5B
(Table 1). Replacement of Thr35
with Asn (T35N), Gln80 with Leu (Q80L), and deletion of the MIR motif of ARL5
were accomplished using a two step PCR technique as described
(Lee et al., 1997). The
5' (sense) mutagenic primer ARL5C and the antisense mutagenic primer
ARL5D were used to generate hARL5(T35N). The 5' (sense) mutagenic primer
ARL5E and the antisense mutagenic primer ARL5F were used to generate
hARL5(Q80L). The point mutation is underlined in oligonucleotide sequences
(Table 1). To generate
ARL5(dMIR) (MIR-deleted mutant) with deletion of 6 amino acids (positions 128
through 133) that include the MIR motif, the 5' (sense) primer ARL5G and
the antisense primer ARL5H were used. To generate ARL5(dC) with deletion of 24
C-terminal amino acids (positions 176 through 201) that include the putative
nuclear localization signal (NLS), hARL5 cDNA clone was digested with
EcoNI and XbaI, blunt-ended, and ligated.
Northern analyses
Blots with RNAs from adult mice and mouse embryos at several stages of
development (Clontech) were processed for hybridization with mARL5-specific
probes as described previously (Lee et
al., 1994).
Expression and purification of recombinant proteins
The entire open reading frame of human ARL5 was obtained by PCR, using
primers that incorporated unique NdeI and BamHI sites,
respectively, at the initiating methionine and six bp downstream from the stop
codon. For preparation of the His-tagged fusion protein, the hARL5 PCR product
was cloned into the expression vector pET15b (Novagen), yielding
pET15b-His-hARL5, which was used to transform BL21 (DE3)
(Lee et al., 1997). Cell
pellets were harvested and His-tagged fusion protein was isolated on
Ni2+-NTA resin (Qiagen, Chatsworth, CA) by standard methods. The
purity of the His-tagged hARL5 was assessed by SDS-PAGE.
Generation of ARL5 antisera and immuno-analyses
Rabbits were immunized with keyhole limpet hemocyanin-conjugated synthetic
peptide GNHLTEMAPTASSFLPC (peptide N), corresponding to the residues 2-18 of
hARL5. Antibodies (ARL5-N) were affinity-purified on immobilized, recombinant
hARL5. Western analysis and immunoprecipitation were performed according to
the procedures of Harlow and Lane (Harlow
and Lane, 1988).
Fractionation by differential centrifugation
Nuclear (N), crude cytosol (C), and membrane (M) fractions were prepared as
described previously (Schreiber et al.,
1989; Yang et al.,
1998
). Briefly, confluent cells were scraped and homogenized in
HES buffer (20 mM HEPES, pH 7.4, 1 mM EDTA, 250 mM sucrose) plus 1 mM
phenylmethylsulfonyl fluoride (PMSF) and a mixture of protease inhibitors
(leupeptin, aprotinin, chymostatin, antipain, and pepstatin, each 1 µg/ml)
at 4°C by 10 strokes in a ball-bearing homogenizer. The cell lysate was
centrifuged at 400 g for 10 minutes to sediment unbroken
cells, nuclei, and cell debris. The supernatant was centrifuged (150,000
g, 1 hour) at 4°C to generate cytosolic (C) and membrane
(M) fractions. To obtain the nuclear fraction, cell pellet containing unbroken
cells, nuclei, and cell debris was dispersed in 1 ml of TBS (Tris-buffered
saline), transferred to an Eppendorf tube and centrifuged for 15 seconds in a
microfuge. TBS was removed and the pellet was suspended in 400 µl of cold
buffer A (10 mM HEPES, pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT,
0.5 mM PMSF) by gentle pipetting in a micro-pipette tip. The cells were
allowed to swell on ice for 15 minutes, after which 25 µl of a solution
containing 10% Nonidet P-40 were added and the tube was vigorously vortexed
for 10 seconds. The homogenate was centrifuged for 30 seconds in a microfuge,
and the nuclear pellet (N) was collected
(Schreiber et al., 1989
).
Equivalent amounts of the nuclear, cytosol and membrane fractions were
analyzed by immunoblotting analysis.
Cell culture and transient transfection
COS-7 cells (ATCC: CRL-1651) were grown in Dulbecco's
modified Eagle's medium (DMEM, GIBCO),
supplemented with 10% fetal bovine serum, 2 mM glutamine and 100 units/ml each
of penicillin and streptomycin. The Hep 3B cell-line (ATCC: HB-8064) was grown
in the same medium with additional 0.1 mM non-essential amino acids. The cells
were subcultured by trypsinization [0.05% (wt/vol) trypsin, with 1% EDTA] and
plating in growth medium in a humidified 5% CO2 incubator at
37°C every 2 to 3 days. The cDNA fragments of hARL5 or its mutants were
fused in-frame to the C-terminus of GFP by subcloning into the EcoRI
and SalI sites of pEGFP-C2 (Clontech). Cells were seeded on
coverslips 16 hours before transfection with the aid of Lipofectamine (Life
Technologies). Freshly prepared solution A (2 µg of plasmid DNA in 50 µl
DMEM) and solution B (6 µl Lipofectamine in 50 µl DMEM) were gently
mixed for 30 minutes at room temperature, added to 400 µl of DMEM and
incubated with cells for 6 hours at 37°C. Additional growth medium with
20% FBS (500 µl) was then added without removing the transfection mixture.
Medium was replaced with fresh growth medium the day after transfection and
cells were harvested 30 to 36 hours later for analysis.
Indirect immunofluorescence staining
Cells were fixed with 4% paraformaldehyde in
PBS-Ca2+-Mg2+ (0.6 mM CaCl2 and 0.5 mM
MgCl2 in PBS) for 15 minutes, incubated with 0.1% Triton X-100 and
0.05% SDS in PBS-Ca2+-Mg2+ for 4 minutes, and in the
same buffer containing 0.2% BSA for an additional 15 minutes, followed by
incubation with primary antibodies [i.e. affinity-purified anti-hARL5-peptide,
mouse anti-p58 (Sigma), mouse anti-ß-COP (Sigma), affinity-purified
rabbit anti-ß-COP peptide (N'-QRKEAADPLASKLNKC-C'), mouse
anti-Flag antibody (M2, Sigma), or mouse anti-C23 (nucleolin, Santa Cruz)] in
the same solution for 40 minutes. After three washes with
PBS-Ca2+-Mg2+, cells were incubated with second
antibody, Alexa 594-conjugated anti-rabbit IgG antibody or Alexa
488-conjugated anti-mouse IgG antibody (Molecular Probes), washed three times
with PBS-Ca2+-Mg2+, mounted on Mowiol (supplemented with
Hoechst 33258), and examined with a Zeiss Axiophot equipped for
epifluorescence according to standard procedures
(Dascher and Balch, 1994).
Primary antibodies, previously depleted of anti-ARL5 activity by incubation
with purified recombinant hARL5, were used as control.
Yeast two-hybrid screen and assay
Yeast strains (L40), plasmids (pBTM116 and pVP16), and library for the
yeast two-hybrid screen were obtained from H. Shih. The genotype of the
Saccharomyces cerevisiae reporter strain L40 is
MATa trp1 leu2 his3 LYS2::lexA-HIS3
URA3::lexA-lacZ (Hollenberg et al.,
1995). Yeast strains were grown at 30°C in rich medium (1%
yeast extract, 2% Bacto-peptone, 2% glucose) or in synthetic minimal medium
with appropriate supplements. PCR fragment of the ARL5, ARL5(Q80L),
ARL5(T35N), ARL5(dMIR), or ARL5(dC) cDNA were generated by using ARL5 and its
mutants as template (as described above) and ARL5I and ARL5B as primers
(Table 1). Plasmids pLexA-ARL5,
pLexA-ARL5(Q79L), pLexA-ARL5(T35N), pLexA-ARL5(dMIR), and pLexA-ARL5(dC),
constructed, respectively, by inserting a PCR-generated fragment of the ARL5,
ARL5(Q80L), ARL5(T34N), ARL5(dMIR), or ARL5(dC) cDNA into the EcoRI
site of the pBTM116 plasmid, were used to express the ARL as a fusion protein
with the DNA-binding domain of LexA as described previously
(Lin et al., 2000
). Control
plasmids pLexA-ARL1, pLexA-ARL3, pLexA-ARL4, pLexA-ARL4(Q79L), and
pLexA-ARL4(T34N) were used and have been described previously
(Lin et al., 2000
).
pACT2-HP1ß, -HP1
, and -HP1
deletion constructs were kindly
provided by Pierre Chambon, Anne Dejean, Jacob Seeler and Howard Worman
(Seeler et al., 1998
;
Nielsen et al., 2001
).
For two-hybrid screening, the yeast reporter strain L40, which contains the
reporter genes lacZ and HIS3 downstream of the binding
sequences for LexA, was transformed with pLexA-ARL5-Q80L and a human liver
pACT2 cDNA library (Clontech) by the lithium acetate method
(Ito et al., 1983), and
subsequently treated as described
(Hollenberg et al., 1995
).
Double transformants were plated with synthetic medium lacking histidine,
leucine, tryptophan, uracil, and lysine. Plates were incubated at 30°C for
three days. His+ colonies were patched on selective plates and
assayed for ß-galactosidase activity by a filter assay
(Hollenberg et al., 1995
).
Plasmid DNA was prepared from colonies displaying a
HIS+/lacZ+ phenotype by electrotransformation
of HB101 cells and used to re-transform the L40 strain containing the
appropriate pLexA-ARL1, pLexA-ARL1(Q71L), pLexA-ARL2, pLexA-ARL3, pLexA-ARL4,
pLexA-ARL4(Q79L), pLexA-ARL4(T34N), pLexA-ARL5, pLexA-ARL5(Q80L),
pLexA-ARL5(T35N), pLexA-ARL5(dC), pLexA-ARL5(dMIR), and pLexA-lamin, to test
for specificity. For assay of ß-galactosidase activity, transformants
were grown in histidine-containing medium, lysed, and assayed as described
(Hollenberg et al., 1995
).
In vitro translation and in vitro interaction
In vitro translation reactions were carried out using the rabbit
reticulocyte lysate TNT system from Promega (Madison. WI). Briefly 1 µg of
DNA was added to a 25-µl TNT reaction containing 12.5 µl of rabbit
reticulocyte lysate, 1 µl of TNT reaction buffer, 10 U of RNA polymerase,
0.5 µl of amino acid mix (minus methionine), 20 µCi of [35S]
methionine and 10 U of RNasin. After incubation at 30°C for 3 hours, 5
µl of the reaction mix were analyzed by SDS-PAGE and autoradiography.
Glutathione-S-transferase (GST) fusion proteins, GST-HP1 or
GST-importin-
, were synthesized in E. coli BL21 by induction
with 0.5 mM isopropyl-D-thiogalactopyranoside (IPTG) at 37°C. Cell pellets
were lysed and subsequently sonicated. GST fusion proteins (
10 µg)
bound to glutathione Sepharose beads (50 µl) in GST-binding buffer [50 mM
KCl, 20 mM HEPES (pH 7.9), 2 mM EDTA, 0.1% Nonidet P-40, 10% glycerol, 0.5%
nonfat dry milk, and 5 mM dithiothreitol] were mixed with 5 µl of in
vitro-translated proteins of interest and incubated at 4°C for 1 hour.
Beads were then washed four times with 1 ml of the same buffer. Bound proteins
were eluted by boiling in 20 µl of 2x protein sample buffer and
separated by SDS-PAGE in 12% gel. The input lane in each experiment contained
10% of the total amount analyzed. GST fusion proteins were stained with
Coomassie blue to evaluate equal loading, and bound proteins were visualized
after autoradiography.
In vivo interaction
COS-7 cells were grown in DMEM with glutamine penicillin/streptomycin
(Gibco) and 10% fetal bovine serum and transfected using Lipofectamine (Life
Tech.). Cell transfected with hARL5(WT), (Q80L), (T35N), (dC), or (dMIR) in
pEGFP-C2 or with Flag-tagged HP1 in pCMV-tag2 were harvested 48 hours
later and proteins detected by western blotting using either a rabbit
polyclonal anti-GFP antibody or a mouse monoclonal anti-Flag antibody (M2,
Sigma). For immunoprecipitation of the HP1
- hARL5 complex, cells were
co-transfected with the appropriate hARL5 construct and HP1
, harvested
48 hours later, washed three times in PBS by centrifugation at 200
g for 10 minutes at 25°C, dispersed in 300 µl of PBS,
and placed on ice. DSP {[dithiobis (succinimidylpropionate)], Pierce} was then
added to a final concentration 1 mg/ml. After 1 hour at 0°C, reaction was
stopped by addition of 1 M Tris-HCl (pH 7.5) to a final concentration of 20-50
mM, and 15 minutes later cells were harvested, washed twice in PBS and once in
binding buffer (TBS: 50 mM Tris-HCl, pH 7, 0.15 M NaCl). Cells were lysed by
sonification in 1 ml of binding buffer containing 1 mM phenylmethylsulfonyl
fluoride plus other protease inhibitors (leupeptin, aprotinin, chymostatin,
antipain, and pepstatin, each 1 µg/ml), and lysates were centrifuged
(10,000 g, 15 minutes). As recommended by the manufacturer, 30
µl of M2 anti-Flag affinity gel (Sigma) were added to the supernatant, and
the resulting mixture was incubated at 4°C for 3 hour with agitation.
After three washes in TBS, beads were dispersed in appropriate amounts of
sample buffer for western blotting analysis of bound proteins using anti-GFP
polyclonal antibody.
Expression and detection of myristoylated ARL5
To produce myristoylated proteins, BL21(DE3) competent bacteria were
co-transformed with pT7-ARL5 and pACYC177/ET3d/yNMT, which encodes yeast
(S. cerevisiae) N-myristoyltransferase
(Haun et al., 1993), and
selected for both ampicillin and kanamycin resistance.
[9,10(n]-3H)myristic acid (1 mCi/ml in ethanol; Amersham) was added
to a final concentration of 30 µCi/ml and the cultures were further treated
as previously described (Huang et al.,
1999
).
Metabolic labeling and immunoprecipitation
COS-7 cells were cultured in Dulbecco's modified Eagle's medium(DMEM)
containing 10% fetal bovine serum and glutamine at 37°C in a humidified 5%
CO2 atmosphere. Cells in 6-well plates (1x105
cells/well) were grown for 16 hours before transfection with 1 µg of
pcDNA3.1A containing hARL5(WT) or hARL5(G2A) mutant cDNA and 7 µl of
LipofectAMINE reagent (Life Technologies) in 1 ml of serum-free DMEM. After
incubation for 5 hours, 1 ml of DMEM containing 20% fetal bovine serum was
added to each well and 36 hours after transfection, cells were incubated for
12 hours with 1 ml of Dulbecco's modified Eagle's medium containing 2% fetal
bovine serum and 150 µCi [9,10-3H]myristic acid (Amersham
Pharmacia Biotech.). The [9,10-3H]myristic acid had been dried
under N2 and dissolved in DMSO. Final concentration of DMSO in the
medium was 0.1%. Cells were washed with PBS and lysed in 2 ml of
radioimmunoprecipitation buffer (50 mM Tris-HCl(pH 8.0), 150 mM NaCl, 1%
Nonidet P-40, 0.5% sodium deoxy cholate, 0.1% SDS, proteinase inhibitors) for
40 minutes at 4°C. Cell lysates were centrifuged (5 minutes, 15,000
g) at room temperature in a microcentrifuge, and supernatants
were collected for immunoprecipitation. Following clearance with protein
A-Sepharose, to each ml of supernatants, 10 µl of anti-myc monoclonal
antibody (9E10) was added. After rocking at 4°C for 6 hours, 30 µl of
protein A-Sepharose was added, and the samples were tumbled overnight at
4°C. Beads were pelleted by centrifugation (30 seconds, 600
g) and washed five times with immunoprecipitation buffer.
Bound proteins were eluted in 20 µl of 2x SDS sample buffer and
separated by SDS-PAGE in 12% gel. Gels were fixed, dried, and exposed to
Hyperfilm for 21 days at -80°C using.
CTA (cholera toxin A subunit)-catalyzed ADP-ribosylation and
nucleotide binding assay
Samples (5 µg) of purified His-tagged hARL5 or yARF1 were tested for
their ability to stimulate cholera toxin-catalyzed auto-ADP-ribosylation
(Huang et al., 1999). Binding
of GTP
S to purified recombinant hARL5 was determined by a filtration
method (Northup et al., 1983
)
with minor modification as previously described
(Lee et al., 1997
).
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Developmentally regulated expression of mARL5
On Northern blot analysis, a 1.4-kb transcript of mARL5 was more
abundant in liver than in other adult mouse tissues
(Fig. 2A). In mouse embryos,
the level of mARL5 mRNA was high on embryonic day 7 and hardly detected by day
11 (Fig. 2B). We have tried,
thus far without success, to localize mARL5 RNA in mouse embryos by in situ
hybridization. This is reminiscent of the low level of ARL5 mRNA in the day-11
embryo, and makes it difficult to establish tissue distribution of mARL5
before day-8 mouse embryo.
|
Subcellular localization of ARL5
To identify the intracellular location of ARL5 protein, we prepared
ARL5-specific antibodies against a unique peptide sequence (residues 2-18) of
human ARL5. The affinity-purified antibodies are sensitive and specific for
detection of both human and mouse ARL5 proteins. Immunoblotting with this
antiserum detected ARL5 in low nanogram amounts, while no reaction was
detected with 100 ng of other recombinant ARLs
(Fig. 3A).
|
To assess the subcellular distribution of ARL5 in human hepatoma Hep3B cells, homogenates were fractionated by differential centrifugation. Nuclear (N), membrane (M), and cytosol (C) fractions were separated and hARL5, PCNA (proliferating cell nuclear antigen), and tubulin (cytoplasmic marker) in subcellular fractions were identified by western blot analysis (Fig. 3B). Endogenous hARL5 was detected in the nuclear fraction (Fig. 3B). Immuno-reactivity with hARL5 was abolished by prior incubation of the antiserum with recombinant hARL5 (data not shown). By immunofluorescence microscopy, endogenous hARL5 in Hep3B and Huh7 cells was distributed mainly over the nucleus (Fig. 3C). No immunoreactivity was detected with preimmune serum or after incubation of antibody ARL5-N with purified recombinant hARL5 (not shown). Nuclei were stained with the DNA-binding dye H33258 (Fig. 3Cb,e) and Golgi with anti-ß-COP antibodies (Fig. 3Cc,f).
Nucleolar localization of ARL5-T35N mutant
To determine whether the subcellular localization of hARL5, like that of
hARL4, was dependent on GTP or GDP binding
(Lin et al., 2000), COS-7
cells transiently expressing GFP-tagged hARL5, hARL5 (Q80L) (predicted to be
GTP-bound), hARL5(T35N) (predicted to be GDP-bound), or hARL5(dC) (lacking the
NLS) were inspected by fluorescence microscopy
(Fig. 4). hARL5 appeared to be
located in nuclei and partially in nucleoli. hARL5(T35N), but not hARL5(Q80L),
appeared to be more concentrated in nucleoli
(Fig. 4, arrows).
Colocalization of hARL5(T35N) with nucleolin, a marker for nucleoli, confirmed
its nucleolar localization. hARL5(dC), not seen in nuclei, distributed, in
part, in a punctate pattern in the cytoplasmic region and, in part,
co-localized with the Golgi marker p58
(Fig. 4, arrowheads). When
subcellular distribution of transiently expressed hARL5 and its mutants in
homogenates of COS-7 cells was examined, hARL5, hARL5(Q80L), and hARL5(T35N)
were detected mainly in the nuclear fraction, with very little in the membrane
fraction (data not shown). hARL5(dC), however, appeared mainly in the membrane
fraction, confirming the results of fluorescence microscopy.
|
Identification of HP1 as a binding partner of ARL5
To identify molecules that might act as down-stream effectors of hARL5, we
used plasmid pLexA-hARL5(Q80L) to express the putatively constitutively active
mutant of hARL5 (hARL5(Q80L)) as bait in a yeast two-hybrid screen of a human
liver cDNA library (Hollenberg et al.,
1995). The bait caused no intrinsic transcriptional activation of
the reporters. Plasmids associated with ß-galactosidase production were
identified from a screen of approximately 4x106 colonies. The
DNA sequence of each library insert was determined and five different inserts
were chosen for further analysis. DNA sequencing and database searches
revealed that nucleotide sequences of three clones encoded human HP1
(accession number L07515), two were full-length and one encoded amino acids
26-191. Thus, the interaction of HP1
with ARL5 appeared not to involve
its N-terminus. Recently, a MIR motif, VPVVVL, has been proposed to be a
consensus sequence for HP1
binding
(Murzina et al., 1999
;
Smothers and Henikoff, 2000
).
Because, a similar sequence, VPVLVL (aa 128-133), is present in ARL5
(Fig. 4A), we constructed a
mutant lacking this motif (hARL5(dMIR)) to assess its interaction with
HP1
. We also used wild type hARL5 and hARL5(T35N) to test whether the
interaction with HP1 is nucleotide-dependent. The LexA-ARL5 fusion proteins
were expressed in yeast and detected by antibodies against LexA or ARL5
(Fig. 5Aa). In the yeast
two-hybrid assay, transformants containing interacting proteins that
transactivate two reporter genes, HIS3 and LacZ, exhibit
ß-galactosidase activity and can grow on minimal medium lacking
histidine. As illustrated in Fig.
5B, LexA-hARL5 and LexA-hARL5(Q80L), but not LexA-hARL5(T35N),
LexA-hARL5(dMIR), LexA-hARL1, LexA-hARL3, LexA-hARL4, or LexA-hARL4(T34N),
interacted with the Gal4AD-HP1
fusion protein and activated the
reporter genes. Interaction of LexA-hARL4(Q79L) with the Gal4AD-HP1
fusion protein was much less than that of LexA-hARL5(Q80L). The
LexA-hARL4(Q79L) and LexA-hARL5(Q80L) were present in relatively equal amounts
in each of the transformed yeast (Fig.
5Ab). At least three HP1 family proteins (HP1
, HP1ß,
and HP1
) have been characterized (reviewed by
Eissenberg and Elgin, 2000
).
Interaction of Gal4AD-HP1ß and Gal4AD-HP1
with the LexA-hARL5(WT)
and LexA-hARL5(Q80L) fusion protein was much less than the HP1
interaction (Fig. 5Ca). The
Gal4AD-HP1
, Gal4AD-HP1ß and Gal4AD-HP1
were also found in
relatively equal amounts in the transformed yeast
(Fig. 5Cb). To identify the
ARL5-interacting domain of HP1
, in the yeast two-hybrid assay
(Fig. 6A), we used four
deletion derivatives, which were expressed in relatively equal amounts
(Fig. 6C). Two regions
(residues 1-58 and 158-175) of HP1
appeared to be important for ARL5
interaction (Fig. 6A,B).
|
|
To confirm that the interactions between hARL5 and HP1 were direct,
hARL5 and its mutants were synthesized in an in vitro translation system and
incubated with GST-HP1
immobilized on glutathione Sepharose. As shown
in Fig. 7A, recombinant
GST-HP1
, but not GST, adsorbed significant amounts of hARL5 and
hARL5(Q80L), whereas no binding of hARL5(T35N) or hARL5(dMIR) was
detected.
|
Interaction of importin- with ARL5 C-terminal nuclear
localization signal NLS
Because hARL5, like hARL4, contains a putative bipartite NLS
(K189RKKAARGGKKRR201) at its C-terminus
(Fig. 4A), we used a hARL5
mutant lacking this sequence, (hARL5(dC)), to test its interaction with
importin-, (karyopherin alpha 2; accession number NM_002266)
(Cuomo et al., 1994
;
Weis et al., 1995
), which had
also been isolated in the two-hybrid screening. LexA-hARL5, LexA-hARL5(Q80L),
and LexA-hARL5(T35N), but not LexA-hARL5(dC), LexA-hARL1, or LexA-hARL3,
interacted with the Gal4AD-importin-
fusion protein and activated the
reporter genes (data not shown). An in vitro GST pull-down assay confirmed
that the interactions between ARL5 and importin-
are direct. hARL5 and
its mutants, produced by in vitro translation, were incubated with immobilized
GST-importin-
in vitro. As shown in
Fig. 7B, recombinant GST-
importin-
, but not GST, adsorbed significant amounts of ARL5,
ARL5(Q80L), and ARL5(T35N), whereas no binding of ARL5(dC) was detected.
Binding of ARL5 and the two mutants (expected to exist largely in GTP-or
GDP-bound forms) was not grossly different.
Intracellular localization and interaction of hARL5 and its mutants
with HP1
To assess the interaction of hARL5 and HP1 in vivo, we compared the
subcellular localization of hARL5 and its mutants with that of HP1
.
COS-7 cells transiently co-expressing Flagtagged HP1
and GFP-tagged
hARL5, hARL5(Q80L), hARL5(T35N), ARL5(dMIR), or ARL5(Q80L/dMIR) were inspected
by fluorescence microscopy (Fig.
8). hARL5(Q80L), and to a lesser extent hARL5, was in part
co-localized with overexpressed HP1
(Fig. 8), and this was
apparently abolished by deletion of MIR motif. ARL5(dMIR) and ARL5(Q80L/dMIR)
apparently, in part, localized to nucleoli
(Fig. 8q,t,u,x). Distribution
of ARL5 and its mutants were similar to those shown in
Fig. 4. hARL5(dC) was, in part,
distributed in a punctate pattern in the cytoplasmic region and, in part,
co-localized with the Golgi marker (data not shown). After subcellular
fractionation, transiently expressed ARL5(dMIR) was detected mainly in the
nuclear fraction with very little in the membrane fraction, confirming the
results of fluorescence microscopy (data not shown).
|
For immunoprecipitation of the HP1-hARL5 complex, cells were
co-transfected with the appropriate hARL5 construct and HP1
. As shown
in Fig. 9, recombinant
GST-HP1
co-immunoprecipitated with hARL5 and hARL5(Q80L), whereas no
association with hARL5(T35N), or hARL5(dMIR) was detected. These data extend
the earlier findings and confirm the importance of the GTP-bound state of the
hARL5(Q80L) and of the MIR motif for formation of a hARL5-HP1
complex
in cells.
|
Biochemical properties of ARL5 protein
The hARL5 fusion protein, like those of hARL2 and hARL3, failed to
stimulate auto-ADP-ribosylation of the cholera toxin A1 protein (data not
shown). hARL5 did bind GTPS in a concentration-dependent manner that
reached a steady state within 60 minutes at 30°C. Phospholipids that
increased GTP
S binding by hARF1 markedly decreased binding by hARL5 as
they bid binding by ARL4 (data not shown).
To determine whether hARL5 could be myristoylated, hARL5, hARL1, and yARL3
were co-expressed in E. coli with yeast N-myristoyltransferase. hARL5
and hARL1 [as reported previously (Lee et
al., 1997)] were myristoylated
(Fig. 10A). yARL3, previously
shown not to be myristoylated (Huang et
al., 1999
), served as a negative control. Because of the low
affinity of our anti-ARL5N peptide antibody, we have failed to isolate
endogenous ARL5 to assess its myristoylation in human cells. We did test
whether overexpressed ARL5 could be myristoylated. COS-7 cells were
transfected with C-terminal Myc-tagged ARL5(WT) and ARL5(G2A), in which Gly at
position two was replaced by Ala. Cells were incubated with
[3H]myristic acid. Immunoprecipitated ARL5(WT), but not ARL5(G2A),
was [3H]-myristoylated (Fig.
10B). Although two forms of ARL5(WT) and ARL5(G2A) with different
mobilities on SDS-PAGE were immunoprecipititated by the anti-Myc antibody,
only the upper band reacted with the anti-hARL5N peptide antibody. We believe
that the lower bands of both ARL5(WT) and ARL5(G2A)
(Fig. 10B, asterisk) were
degraded at the N-terminus, and react, therefore, with anti-Myc, but not
anti-hARL5N, antibodies. It appears that the biological function of ARL5, like
those of ARL1, ARL4, the ARFs, and other proteins, may be influenced by
myristoylation.
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Although the role of the nucleolus as a factory for assembling ribosomal
subunits is well established, one of the more interesting findings is the
presence thereof a variety of macromolecules that have no apparent ribosomal
function (reviewed by Garcia and Pillus,
1999; Olson et al.,
2000
). The nucleolus seems to have roles also in assembling
ribonucleoprotein (RNP), modifying small RNAs, controlling aging, sequestering
regulatory molecules, and nuclear export. In these events, the nucleolus
serves as a site for both recruitment and exclusion of regulatory complexes.
Recently, several cell-cycle regulators whose activity is controlled by
sequestration in the nucleolus have been identified (reviewed by
Visintin and Amon, 2000
). To
our knowledge, ARL5 and ARL4 may be the first subfamily of small GTPases
reported to be localized in nucleoli in a manner dependent on GDP binding.
Studies of nuclear architecture reveal that the dynamic properties of nuclear
proteins are critical for their function (reviewed by
Misteli, 2001
). The mobility
of proteins facilitates their availability throughout the nucleus, and their
dynamic interplay generates an everchanging, but overall stable, architectural
framework, within which nuclear processes take place. Overall, nuclear
morphology is determined by the functional interactions of nuclear components.
The dynamic properties of nuclear proteins are consistent with a central role
for stochastic mechanisms in gene expression and nuclear architecture.
Although the physiological function of ARL5 and ARL4 in nuclei and nucleoli is
not understood, it will be important to determine whether ARL5 and ARL4
participate in the regulation of nuclear protein dynamics via their nucleolar
sequestration.
Of five proteins that interacted with hARL5 in the yeast two-hybrid
screening, one that interacted with ARL5(Q80L) and ARL5(WT), but not
ARL5(T35N) or ARL5(dMIR), in the two-hybrid system is a known HP1.
HP1
proteins are believed to represent important structural components
of heterochromatin, consistent with their involvement in position-effect
variegation (reviewed by Singh,
1994
; Elgin,
1996
), as well in proper functioning of the centromere during
mitosis (Kellum and Alberts,
1995
). HP1 proteins are phosphorylated in vivo and this
phosphorylation may be important mechanism for regulating their
multimerization and/or other interactions. HP1
, HP1ß, and
HP1
share two highly conserved globular domains, an N-terminal chromo
domain (CD) and a C-terminal chromo shadow domain (CSD), which span residues
22-66 and 123-175 of HP1
, respectively
(Eissenberg and Elgin, 2000
).
The CD and CSD of HP1 family are connected by a poorly conserved hinge region
(H). Several cellular proteins have been reported to interact directly with
HP1 proteins through the CSD; these include the lamin B receptor
(Ye et al., 1997
), the
transcriptional cofactors TIF1 ß and other chromosomal regulators
(Le Douarin et al., 1996
), the
chromatin assembly factor 1 (CAF-1) subunit p150
(Murzina et al., 1999
), as
well as PML/SP100 nuclear bodies (Seeler
et al., 1998
). Both CSD and CD domains of HP1 are required for
interaction with ORC (Pak et al.,
1997
). HP1
, via its CD, also binds to histone H3,
suggesting a role for histone H3 in anchoring CD-containing proteins to the
chromatin fiber (Nielsen et al.,
2001
). Interactions of HP1
with SWI/SNF complex member
BRG-1 and with a RAD-54- like protein called HP-BP-38 also have been reported
(Le Douarin et al., 1996
).
Although the precise functional consequences of these interactions, or of the
interaction of ARL5 with HP1
, remain to be determined, these data
suggest that HP1
proteins might represent important targets for
cellular regulators of DNA transcription, replication, or repair.
Several proteins, such as the p150 subunits of CAF-1, TIF1, and
TIFß, bound to HP1
directly through an amino-terminal sequence,
termed MIR for MOD1 (mouse HP1
)-interaction
region (Murzina et al.,
1999
). Mutations of MIR prevented p150 from binding to HP1
proteins (Murzina et al.,
1999
). The in vitro and in vivo experiments using GTPase-defective
ARL5(Q80L) and GTP-binding-defective ARL5(T35N) mutants of ARL5, as well as
ARL5-dMIR, demonstrated GTP-dependent interaction of hARL5 with HP1
that requires its MIR-like motif. All three clones from yeast two-hybrid
screening, two full-length and one encoding amino acids 26-191, interacted
strongly with ARL5(WT) and ARL5(Q80L), suggesting that the interaction did not
involve the N-terminal 1-25 amino acids of HP1
. In the yeast two-hybrid
interaction, two regions (residues 1-58 and 158-175) of HP1
were
important for ARL5 interaction. Thus, we infer that the interaction of
HP1
with ARL5, similar to that of ORC
(Pak et al., 1997
), involves
parts of both the CD and CSD regions. Unlike other ARLs, ARL4 also contains a
MIR-like motif: 127VPVLIV132. However, interaction of
hARL4(Q79L) with HP1
is, apparently, much weaker than that of
ARL5(Q80L). Three-dimensional structures of mouse HP1 CD and CSD have been
published (Ball et al., 1997
;
Brasher et al., 2000
), showing
that proteins containing the MIR motif bind to the CSD when HP1 is in a
dimeric form (Brasher et al.,
2000
). Thus, it is conceivable that other domain(s) of ARL5 might
also be involved in interacting with HP1 through its CD and CSD regions. ARL5
interacted with all three HP1 paralogues, although the lower binding of
HP1ß and HP1
in the two-hybrid assay does not necessarily mean
that this also occurs in a mammalian cell context. The HP1 isotype-specificity
of ARL5 need to be evaluated further in vivo, during both interphase and
mitosis.
The MIR-like motif, 128VPVLVL133, in ARL5, is
adjacent to the highly conserved 135NKQD138 sequence,
which is directly involved in the binding of the guanine nucleotide base.
Intriguingly, like ARL5(T35N), ARL5(Q80L/dMIR), in part, is localized to
nucleoli, although most of ARL5(Q80L) is found in nucleoplasm, in part
associated with HP1 (Fig.
8). It will be important to learn whether deletion of the MIR-like
motif from ARL5(Q80L) either directly alters its localization domain(s) or
changes the GTP-bound to a GDP-bound conformation, with subsequent
localization to nucleoli. Further structural characterization of ARL5 should
help to reveal the impact of deletions on the ARL5 folding, perhaps explaining
how removal of the MIR-like motif prevents interaction of ARL5 with HP1 and
affects subcellular localization of ARL5(Q80L).
Two of the proteins that interacted with ARL5 have been localized to the
cytoplasm. One of these is a known GEF (cytohesin-2/ARNO, accession number
U70728) for ARF [(Frank et al.,
1998; Macia et al.,
2001
) C.-C. Lee, C.-Y. Lin, J.-C. Kuo and F.-J.S.L., unpublished]
and it will be interesting to learn whether it can translocate into nuclei or
can activate ARL5. Alternatively, hARL5 may be cytoplasmic, as was hARL5-dC,
which had a Golgi-like or punctate distrbution. The small GTPase Ran, which
plays a key role in nuclear transport, also functions in mitosis by regulating
microtubule nucleation and/or growth
(Heald and Weis, 2000
).
Recently, Ran, in concert with importin
and ß, was reported to
regulate spindle formation (Gruss et al.,
2001
; Nachury et al.,
2001
). The nuclear envelope of higher eukaryotes is a dynamic
structure that breaks down during prometaphase, reforming during anaphase and
telophase (Gant and Wilson,
1997
). During nuclear envelope breakdown, nuclear lamina and pore
complexes disassemble, and nuclear membranes vesiculate. During reassembly,
nuclear membranes associated with daughter chromosomes fuse to enclose the
chromatin. The nucleus then enlarges by importation of proteins through newly
assembled pore complexes and fusion of additional vesicles. HP1 could serve as
a linker, connecting peripheral heterochromatin to the inner nuclear membrane
and mediating nuclear envelope reassembly at the end of mitosis
(Ye et al., 1997
;
Kourmouli et al., 2000
). A
requirement for a non-ARF GTPase for nuclear fusion and mitotic membrane
disassembly was suggested (Gant and
Wilson, 1997
). We speculate that activated GTP-bound ARL5 may be
recruited by HP1 to the heterochromatin regions and have a role in novel
nuclear membrane dynamics. It was also suggested that phosphorylation of HP1
proteins can alter their function in the cell cycle and/or development
(Zhao and Eissenberg, 1999
).
Clearly, further studies are necessary to determine the precise role of the
interaction between hARL5 and HP1
in embryonic development. To gain new
insight into the physiological function of ARL5, we are generating a mouse
strain with a targeted deletion of ARL5-coding sequence. We are, of course,
interested to determine whether embryonic development is impaired in the ARL5
knock-out mice. These animals should facilitate the identification of
additional physiological role(s) of ARL5.
![]() |
Acknowledgments |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Ball, L. J., Murzina, N. V., Broadhurst, R. W., Raine, A. R.,
Archer, S. J., Stott, F. J., Murzin, A. G., Singh, P. B., Domaille, P. J. and
Laue, E. D. (1997) Structure of the chromatin binding
(chromo) domain from mouse modifier protein 1. EMBO J.
16,2473
-2481.
Bobak, D. A., Nightingale, M. S., Murtagh, J. J., Price, S. R., Moss, J. and Vaughan, M. (1989). Molecular cloning, characterization, and expression of human ADP-ribosylation factors: two guanine nucleotide-dependent activators of cholera toxin. Proc. Natl. Acad. Sci. USA 86,6101 -6105.[Abstract]
Boman, A. L. and Kahn, R. A. (1995). Arf proteins: the membrane traffic police? Trends Biochem. Sci. 20,147 -150.[CrossRef][Medline]
Brasher, S. V., Smith, B. O., Fogh, R. H., Nietlispach, D.,
Thiru, A., Nielsen, P. R., Broadhurst, R. W., Ball, L. J., Murzina, N. V. and
Laue, E. D. (2000) The structure of mouse HP1 suggests a
unique mode of single peptide recognition by the shadow chromo domain dimer.
EMBO J. 19,1587
-1597.
Cavenagh, M. M., Breiner, M., Schurmann, A., Rosenwald, A. G.,
Terui, T., Zhang, C.-j., Randazzo, P. A., Adams, M., Joost, H.-G. and Kahn, R.
A. (1994). ADP-ribosylation factor (ARF)-like 3, a new member
of the ARF family of GTP-binding proteins cloned from human and rat tissues.
J. Biol. Chem. 269,18937
-18942.
Clark, J., Moore, L., Krasinskas, A., Way, J., Batttey, J., Tamkun, J. and Kahn, R. A. (1993). Selective amplification of additional members of the ADP-ribosylation factor (ARF) family: cloning of additional human and Drosophila ARF-like genes. Proc. Natl. Acad. Sci. USA 90,8952 -8956.[Abstract]
Cuomo, C. A., Kirch, S. A., Gyuris, J., Brent, R. and Oettinger, M. A. (1994). Rch1, a protein that specifically interacts with the RAG-1 recombination-activating protein. Proc. Natl. Acad. Sci. USA 91,6156 -6160.[Abstract]
Dascher, C. and Balch, W. E. (1994). Dominant
inhibitory mutants of ARF1 block endoplasmic reticulum to Golgi transport and
trigger disassembly of the Golgi apparatus. J. Biol.
Chem. 269,1437
-1448.
Eissenberg, J. C. and Elgin, S. C. (2000). The HP1 protein family: getting a grip on chromatin. Curr. Opin. Genet. Dev. 10,204 -210.[CrossRef][Medline]
Eissenberg, J. C., James, T. C., Foster-Harnett, D. M., Harnett, T., Ngan, V. and Elgin, S. C. (1990). Mutation in a heterochromatin-specific chromosomal protein is associated with suppression of position-effect variegation in Drosophila melanogaster. Proc. Natl. Acad. Sci. USA 87,9923 -9927.[Abstract]
Elgin, S. C. (1996). Heterochromatin and gene regulation in Drosophila. Curr. Opin. Genet. Dev. 6, 193-202.[CrossRef][Medline]
Fanti, L., Giovinazzo, G., Berloco, M. and Pimpinelli, S. (1998). The heterochromatin protein 1 prevents telomere fusions in Drosophila. Mol. Cell 2, 527-538.[Medline]
Frank, S., Upender, S., Hansen, S. H. and Casanova, J. E.
(1998). ARNO is a guanine nucleotide exchange factor for
ADP-ribosylation factor 6. J. Biol. Chem.
273, 23-27.
Gant, T. M. and Wilson, K. L. (1997). Nuclear assembly. Annu. Rev. Cell Dev. Biol. 13,669 -695.[CrossRef][Medline]
Garcia, S. N. and Pillus, L. (1999). Net results of nucleolar dynamics. Cell 97,825 -828.[Medline]
Gruss, O. J., Carazo-Salas, R. E., Schatz, C. A., Guarguaglini, G., Kast, K., Wilm, M., le Bot, N., Vernos, I., Karsenti, E. and Mattaj, I. W. (2001). Ran induces spindle assembly by reversing the inhibitory effect of importin alpha on TPX2 activity. Cell 104,83 -93.[Medline]
Harlow, E. and Lane, D. (1988).Antibodies: A Laboratory Manual . Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press.
Haun, R. S., Tsai, S.-C., Adamik, R., Moss, J. and Vaughan,
M. (1993). Effect of myristoylation on GTP-dependent binding
of ADP-ribosylation factor to Golgi. J. Biol. Chem.
268,7064
-7068.
Heald, R. and Weis, K. (2000). Spindles get the ran around. Trends Cell Biol. 10, 1-4.[CrossRef][Medline]
Hollenberg, S. M., Sternglanz, R., Cheng, P. F. and Weintraub, H. (1995). Identification of a new family of tissue-specific basic helix-loop-helix proteins with a two-hybrid system. Mol. Cell. Biol. 15,3813 -3822.[Abstract]
Hong, J.-X., Lee, F.-J. S., Patton, W. A., Lin, C.-Y., Moss, J.
and Vaughan, M. (1998). Phospholipid- and GTP-dependent
activation of cholera toxin and phospholipase D by human ADP-ribosylation
factor-like protein 1 (HARL1). J. Biol. Chem.
273,15872
-15876.
Huang, C.-F., Buu, L.-M., Yu, W.-L. and Lee, F.-J. S.
(1999). Characterization of a novel ADP-ribosylation factor-like
protein (yARL3) in Saccharomyces cerevisiae. J. Biol.
Chem. 274,3819
-3827.
Ito, H., Fukuda, Y., Murata, K. and Kimura, A. (1983). Transformation of intact yeast cells treated with alkali cations. J. Bacteriol. 153,163 -168.[Medline]
Jacobs, S., Schilf, C., Fliegert, F., Koling, S., Weber, Y., Schurmann, A. and Joost, H.-G. (1999). ADP-ribosylation factor (ARF)-like 4, 6, and 7 represent a subgroup of the ARF family characterization by rapid nucleotide exchange and a nuclear localization signal. FEBS Lett. 456,384 -388.[CrossRef][Medline]
James, T. C. and Elgin, S. C. (1986). Identification of a nonhistone chromosomal protein associated with heterochromatin in Drosophila melanogaster and its gene. Mol. Cell. Biol. 6,3862 -3872.[Medline]
Kellum, R. and Alberts, B. M. (1995).
Heterochromatin protein 1 is required for correct chromosome segregation in
Drosophila embryos. J. Cell Sci.
108,1419
-1431.
Kourmouli, N., Theodoropoulos, P. A., Dialynas, G., Bakou, A.,
Politou, A. S., Cowell, I. G., Singh, P. B. and Georgatos, S. D.
(2000). Dynamic associations of heterochromatin protein 1 with
the nuclear envelope. EMBO J.
19,6558
-6568.
Le Douarin, B., Nielsen, A. J., Garnier, J. M., Ichinose, H., Jeanmougin, F., Losson, R. and Chambon, P. (1996). A possible involvement of TIF1 alpha and TIF1 beta in the epigenetic control of transcription by nuclear receptors. EMBO J. 15,6701 -6715.[Abstract]
Lee, F.-J. S., Stevens, L. A., Kao, Y. L., Moss, J. and Vaughan,
M. (1994). Characterization of a glucose-repressible
ADP-ribosylation factor 3 (ARF3) from Saccharomyces cerevisiae. J.
Biol. Chem. 269,20931
-20937.
Lee, F.-J. S., Huang, C.-F., Yu, W.-L., Buu, L.-M., Lin, C.-Y.,
Huang, M.-C., Moss, J. and Vaughan, M. (1997).
Characterization of an ADP-ribosylation factor-like 1 protein in Saccharomyces
cerevisiae. J. Biol. Chem.
272,30998
-31005.
Lin, C.-Y., Huang, P.-H., Liao, W.-L., Cheng, H.-J., Huang,
C.-F., Kuo, J. C., Patton, W. A., Massenburg, D., Moss, J. and Lee, F.-J.
S. (2000). ARL4, an ARF-like protein that is developmentally
regulated and localized to nuclei and nucleoli. J. Biol.
Chem. 275,37815
-37823.
Lorentz, A., Ostermann, K., Fleck, O. and Schmidt, H. (1994). Switching gene swi6, involved in repression of silent mating-type loci in fission yeast, encodes a homologue of chromatin-associated proteins from Drosophila and mammals. Gene 143,139 -143.[CrossRef][Medline]
Lowe, S. L., Wong, S. H. and Hong, W. (1996).
The mammalian ARF-like protein 1 (Arl1) is associated with the Golgi complex.
J. Cell Sci. 109,209
-220.
Macia, E., Chabre, M. and Franco, M. (2001).
Specificities for the small G proteins ARF1 and ARF6 of the guanine nucleotide
exchange factors ARNO and EFA6. J. Biol. Chem.
276,24925
-24930.
Misteli, T. (2001). Protein dynamics:
implications for nuclear architecture and gene expression.
Science 291,843
-847.
Moroianu, J., Blobel, G. and Radu, A. (1996).
The binding site of karyopherin alpha for karyopherin beta overlaps with a
nuclear localization sequence. Proc. Natl. Acad. Sci.
USA 93,6572
-6576.
Moss, J. and Vaughan, M. (1995). Structure and
function of ARF proteins: activators of cholera toxin and critical components
of intracellular vesicular transport processes. J. Biol.
Chem. 270,12327
-12330.
Moss, J. and Vaughan, M. (1998). Molecules in
the ARF orbit. J. Biol. Chem.
273,21431
-21434.
Murzina, N., Verreault, A., Laue, E. and Stillman, B. (1999). Heterochromatin dynamics in mouse cells: interaction between chromatin assembly factor 1 and HP1 proteins. Mol. Cell 4,529 -540.[Medline]
Nachury, M. V., Maresca, T. J., Salmon, W. C., Waterman-Storer, C. M., Heald, R. and Weis, K. (2001). Importin beta is a mitotic target of the small GTPase Ran in spindle assembly. Cell 104,95 -106.[Medline]
Nielsen, A. L., Oulad-Abdelghani, M., Ortiz, J. A., Remboutsika, E., Chambon, P. and Losson, R. (2001). Heterochromatin formation in mammalian cells: interaction between histones and HP1 proteins. Mol. Cell 7,729 -739.[CrossRef][Medline]
Northup, J. K., Sternweis, P. C. and Gilman, A. G.
(1983). The subunits of the stimulatory regulatory component of
adenylate cyclase. Resolution, activity, and properties of the 35,000-dalton
(beta) subunit. J. Biol. Chem.
258,11361
-11368.
Olson, M. O., Dundr, M. and Szebeni, A. (2000). The nucleolus: an old factory with unexpected capabilities. Trends Cell Biol. 10,189 -196.[CrossRef][Medline]
Pak, D. T., Pflumm, M., Chesnokov, I., Huang, D. W., Kellum, R., Marr, J., Romanowaski, P. and Botchan, M. R. (1997). Association of the origin recognition complex with heterochromatin and HP1 in higher eukaryotes. Cell 91,311 -323.[Medline]
Sanger, F., Nicklen, S. and Coulson, A. R. (1977). DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74,5463 -5467.[Abstract]
Saunders, W. S., Chue, C., Goebl, M., Craig, C., Clark, R. F.,
Powers, J. A., Eissenberg, J. C., Elgin, S. C. R., Rothfield, N. F. and
Earnshaw, W. C. (1993). Molecular cloning of a human
homologue of Drosophila heterochromatin protein HP1 using anti-centromere
autoantibodies with anti-chromo specificity. J. Cell
Sci. 104,573
-582.
Schreiber, E., Matthias, P., Muller, M. M. and Schaffner, W. (1989). Rapid detection of octamer binding proteins with `mini-extracts', prepared from a small number of cells. Nucleic Acids Res. 17,6419 .[Medline]
Schurmann, A., Breiner, M., Becker, W., Huppertz, C.,
Kainulainen, H., Kentrup, H. and Joost, H.-G. (1994). Cloning
of two novel ADP-ribosylation factor-like proteins and characterization of
their differential expression in 3T3-L1 cells. J. Biol.
Chem. 269,15683
-15688.
Seeler, J.-S., Marchio, A., Sitterlin, D., Transy, C. and
Dejean, A. (1998). Interaction of SP100 with HP1 proteins: a
link between the promyelocytic leukemia-associated nuclear bodies and the
chromatin compartment. Proc. Natl. Acad. Sci. USA
95,7316
-7321.
Singh, P. B. (1994). Molecular mechanisms of
cellular determination: their relation to chromatin structure and parental
imprinting. J. Cell Sci.
107,2653
-2668.
Singh, P. B., Miller, J. R., Pearce, J., Kotharty, R., Burton, R. D., Paro, R., James, T. C. and Gaunt, S. J. (1991). A sequence motif found in a Drosophila heterochromatin protein is conserved in animals and plants. Nucleic Acids Res. 19,789 -794.[Abstract]
Smith, S. A., Holik, P. R., Stevens, J., Melis, R., White, R. and Albertsen, H. (1995). Isolation and mapping of a gene encoding a novel human ADP-ribosylation factor on chromosome 17q12-q21. Genomics 28,113 -115.[CrossRef][Medline]
Smothers, J. F. and Henikoff, S. (2000). The HP1 chromo shadow domain binds a consensus peptide pentamer. Curr. Biol. 10,27 -30.[CrossRef][Medline]
Visintin, R. and Amon, A. (2000). The nucleolus: the magician's hat for cell cycle tricks. Curr. Opin. Cell Biol. 12,372 -377.[CrossRef][Medline]
Weis, K., Mattaj, I. W. and Lamond, A. I. (1995). Identification of hSRP1 alpha as a functional receptor for nuclear localization sequences. Science 268,1049 -1054.[Medline]
Yang, C. Z., Heimberg, H., D'Souza-Schorey, C., Mueckler, M. M.
and Stahl, P. D. (1998). Subcellular distribution and
differential expression of endogenous ADP-ribosylation factor 6 in mammalian
cells. J. Biol. Chem.
273,4006
-4011.
Ye, Q., Callebaut, I., Pezhman, A., Courvalin, J. C. and Worman,
H. J. (1997). Domain-specific interactions of human HP1-type
chromodomain proteins and inner nuclear membrane protein LBR. J.
Biol. Chem. 272,14983
-14989.
Zhang, G.-F., Patton, W. A., Lee, F.-J. S., Liyanage, M., Han,
J.-S., Rhee, S. G., Moss, J. and Vaughan, M. (1995).
Different ARF domains are required for the activation of cholera toxin and
phospholipase D. J. Biol. Chem.
270, 21-24.
Zhao, T. and Eissenberg, J. C. (1999)
Phosphorylation of heterochromatin protein 1 by casein kinase II is required
for efficient heterochromatin binding in Drosophila. J. Biol.
Chem. 274,15095
-15100.