XMog1, a nuclear Ran-binding protein in Xenopus, is a functional homologue of Schizosaccharomyces pombe Mog1p that co-operates with RanBP1 to control generation of Ran-GTP
Francisco J. Nicolás1,*,
William J. Moore1,
Chuanmao Zhang1,2 and
Paul R. Clarke1,
1
Biomedical Research Centre, University of Dundee, Level 5, Ninewells Hospital
and Medical School, Dundee, DD1 9SY, UK
2
Department of Cell Biology and Genetics, College of Life Sciences, Peking
University, Beijing 100871, China
*
Present address: ICRF Laboratories, 44 Lincoln's Inn Fields, London, WC2A 3PX,
UK
Author for correspondence (e-mail:
p.clarke{at}icrf.icnet.uk
)
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Fig. 1. Sequence of Xenopus laevis Mog1 (XMog1). Comparison of the amino
acid sequence of XMog1 (GenBank/EMBL/DDBJ database accession number AJ278788)
with related sequences from human (AF161514), Arabidopsis thaliana
(AC013289), Schizosaccharomyces pombe (AL031179) and
Saccharomyces cerevisiae (P47123).
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Fig. 2. Two-hybrid interaction between XMog1 and Ran. The growth of yeast on plates
with (His+) or without (His-) histidine is shown. Yeast
were co-transformed with a plasmid encoding the activation domain of Gal4
fused to XMog1 (pAD-XMog1) and a second plasmid encoding the DNA-binding
domain of GAL4 (pDB) fused to wild-type Ran, RanQ69L, RanT24N or p53 as a
negative control. Co-transformation with Gal4AD/SV40 large T antigen and
Gal4BD/p53 was used as a positive control. The diagram illustrates the
position of each co-transformant on the plates.
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Fig. 3 Analysis of mog1 in Schizosaccharomyces pombe. (A) Tetrad
analysis of FJN1, a strain in which one genomic copy of mog1 is
replaced with a KanR cassette. Two out of the four haploid
spores in each column germinated, indicating a requirement of mog1
for viability. (B) Complementation analysis in S. pombe. Growth of
colonies derived from germinated spores carrying  mog1
transformed with plasmids encoding S. pombe Mog1p (SpMog1), S.
cerevisiae Mog1p (ScMog1) or Xenopus laevis Mog1 (XMog1) under
the control of the thiamine-repressible nmt1 promoter.
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Fig. 4. Cellular localisation of XMog1. (A) Western blot showing reactivity of a
purified polyclonal antibody against XMog1, GST-XMog1, Xenopus egg
extract (XEE) and Xenopus somatic XTC cell extract (XTC). (B)
Xenopus XTC cells were labelled with purified antibodies directed
against XMog1 and counterstained with DAPI to reveal the distribution of DNA.
(C) Cos-1, Hela and 3T3 cells were transfected with a fusion of GFP-XMog1. The
cells were fixed 24 hours after the transfection and were counterstained with
DAPI to reveal the distribution of DNA.
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Fig. 5. Ran binds to XMog1 in co-precipitation assays. (A). Binding of XMog1 to
GST-Ran. Incubations were carried out using 2 µM GST-Ran and 0.07 µM
XMog1 purified proteins in Mg2+-buffer with addition of GTP, GDP or
EDTA. A western blot of the precipitates was developed using antibodies to GST
(to show the extent of recovery of GST-Ran) and XMog1. (B) Co-precipitation of
XMog1 from Xenopus egg extract (XEE) by RanQ69L-GTP, RanT24N-GDP or
wild-type Ran-GDP proteins (5 µM) produced as GST-fusions. GST-Ran proteins
were incubated in XEE and recovered on glutathione-Sepharose beads in
Mg2+-buffer. A western blot of the precipitates was developed using
antibodies to GST (to show the extent of recovery of GST-Ran) and XMog1. (C)
Co-precipitation of Ran from XEE by GST-XMog1 with the addition of GTP S
or GDP. A western blot of the precipitates was developed using an antibody to
Ran. Total Xenopus egg extract (XEE) was also loaded.
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Fig. 6. XMog1 alone promotes release of GTP, but not GDP, from Ran. (A) Nucleotide
release in the presence of 1 mM free GDP in exchange buffer containing 10 mM
Mg2+. Ran was loaded with [3H]GTP (open squares) or
[3H]GDP (closed squares). (B) Nucleotide release in the absence of
free nucleotide in exchange buffer containing 10 mM Mg2+. Wild-type
Ran (squares) or RanQ69L (triangles) were preloaded with [3H]GTP
(open symbols) or [3H]GDP (closed symbols). (C) GTP release by
XMog1 in buffers containing 1 mM Mg2+ (closed symbols) or with
addition of 10 mM EDTA (open symbols) in the absence of free nucleotide. For
each datum, Ran (50 pmol) was loaded with the specified radionucleotide and
incubated at 21°C in a total volume of 50 µl with the indicated amount
of XMog1 in exchange buffer (see Materials and Methods). In (C),
Ran-[3H]GTP was prepared with the addition of a lower concentration
of Mg2+ so that the reactions contained 1 mM Mg2+ with
or without the addition of 10 mM EDTA. After incubation for 10 minutes, the
radioactivity remaining bound to Ran was determined as described in Materials
and Methods.
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Fig. 7. XMog1 and RanBP1 together promote GDP release from Ran and loading with
GTP. (A) Binding between Ran (68 nM) and GST-RanBP1 (2.6 µM) in
Mg2+-buffer with addition of GTPS or GDP. Incubations were
carried out with or without addition of 6 µM XMog1. A western blot of the
precipitates was developed using antibodies to Ran and XMog1. (B)
[3H]GTP release from Ran induced by XMog1 is inhibited by RanBP1.
(C) [3H]GDP release from Ran is promoted by XMog1 and RanBP1. In
both (A) and (B), Ran (50 pmol) preloaded with [3H]-labelled
guanine nucleotide was incubated in a total volume of 50 µl with the
specified amount of XMog1 alone (open symbols) or with the addition of XMog1
and 18 pmol RanBP1 (closed symbols) in the absence of free nucleotide, as in
Fig. 6. The amount of
radioactivity remaining was counted and the amount released expressed as a
percentage of the total. (D) [3H]GTP loading onto Ran in the
presence of XMog1 and RanBP1. 50 pmol Ran-GDP was incubated with no additions
(open squares), 300 pmol XMog1 alone (closed squares), 18 pmol RanBP1 alone
(open triangles) or XMog1 and RanBP1 together (closed triangles) in a total
volume of 50 µ1. [3H]GTP was added at 1.2 µM and GDP was
present at 700 µM. Incubation was carried out for the times shown before
determining the amount of radioactivity bound to Ran. Each time point was
performed in duplicate.
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Fig. 8. Effect of XMog1 and RanBP1 on RCC1-mediated guanine nucleotide release. Ran
(50 pmol) preloaded with [3H]GDP was incubated with 1.8 pmol RCC1
for the specified time in a reaction volume of 50 µl containing 700 µM
GDP (A) or 700 µM GTP (B) before determining the amount of radioactivity
remaining bound to Ran. Additions were buffer alone (open squares), 300 pmol
XMog1 (closed squares), 18 pmol RanBP1 (open triangles) or XMog1 and RanBP1
together (closed triangles).
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Fig. 9. Effect of XMog1 and RanBP1 on loading of Ran with guanine nucleotides by
RCC1. (A) Ran (50 pmol) preloaded with unlabelled GTP was incubated with
[3H]GDP for 10 minutes with addition of the specified amount of
RCC1, either without XMog1 (open symbols) or with 300 pmol XMog1 (closed
symbols). (B) Ran (50 pmol) preloaded with unlabelled GTP was incubated with
[3H]GDP for the specified time with 1.8 pmol RCC1 and further
additions of buffer (open squares), 300 pmol XMog1 (closed squares), 18 pmol
RanBP1 (open triangles), or XMog1 and RanBP1 together (closed triangles). (C)
Ran (50 pmol) preloaded with unlabelled GDP was incubated with
[3H]GTP for the 10 minutes with addition of the specified amount of
RCC1 and further additions of buffer (crosses), 300 pmol XMog1 (open circles),
18 pmol RanBP1 (open squares), or XMog1 and RanBP1 together (closed squares).
In all assays the reaction volume was 50 µl.
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Fig. 10. A model for the action of Mog1-related proteins and RanBP1 on guanine
nucleotide exchange on Ran. When RanBP1 is present, Mog1 destabilises GDP
binding to Ran to form a transient, nucleotide-free complex. RanBP1 promotes
GTP loading by stabilising the GTP-conformation. RCC1 catalyses guanine
nucleotide exchange by increasing the rate at which equilibrium is acheived.
Thus, Mog1 and RanBP1 together alter the equilibrium of the reaction to favour
the formation of Ran-GTP.
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© The Company of Biologists Ltd 2001
