Human RagA and RagB is reported to be 52% identical to a putative GTPase of Saccharomyces cerevisiae, Gtr1p. According to the reported nucleotide sequence, we amplified human RagA and RagBs cDNAs from the human B cell cDNA library with PCR. Both cDNAs rescued a cold sensitivity of S. cerevisiae, gtr1-11. Furthermore, we introduced into the cloned human RagA cDNA, the mutation ‘T21L’ corresponding to the gtr1-11 mutation which has been reported to suppress not only all of rcc1-, temperature-sensitive mutants of Ran/Gsp1p GTPase GDP/GTP-exchanging factor, but also rna1-1, a temperature-sensitive mutant of Ran/Gsp1p GTPase-activating protein. The resulting RagAgtr1-11 cDNA partially, but significantly, suppressed both rcc1- and rna1-1 mutations. These results indicated that RagA and RagBs are functional homologues of S. cervisiae Gtr1p. Interestingly, while wild-type human RagA and RagBs were localized within the cytoplasm, similar to S. cerevisiae Gtr1p, the mutated human RagAgtr1-11 corresponding to a dominant negative form of RagA was distributed in discrete speckles in the nucleus, being localized side by side with SC-35, a non-snRNP of the splicing complex. In contrast, a dominant positive form of RagA, Q66L was localized in the cytoplasm. Thus, RagA was suggested to shuttle between the cytoplasm and the nucleus, depending on the bound nucleotide state.

REFERENCES

Aebi
M.
,
Clark
M. W.
,
Vijayraghavan
U.
,
Abelson
J.
(
1990
).
A yeast mutant, PRP20, altered in mRNA metabolism and maintenance of the nuclear structure, is defective in a gene homologous to the human gene RCC1 which is involved in the control of chromosome condensation.
Mol. Gen. Genet
224
,
72
80
Belhumeur
P.
,
Lee
A.
,
Tam
R.
,
DiPaolo
T.
,
Fortin
N.
,
Clark
M. W.
(
1993
).
GSP1 and GSP2, genetic suppressors of the prp20-1 mutant in Saccharomyces cerevisiae: GTP-binding proteins involved in the maintenance of nuclear organization.
Mol. Cell. Biol
13
,
2152
2161
Bischoff
F. R.
,
Ponstingl
H.
(
1991
).
Mitotic regulator protein RCC1 is complexed with a nuclear ras-related polypeptide.
Proc. Nat. Acad. Sci. USA
88
,
10830
10834
Bischoff
F. R.
,
Ponstingl
H.
(
1991
).
Catalysis of guanine nucleotide exchange on Ran by the mitotic regulator RCC1.
Nature
354
,
80
82
Bischoff
F. R.
,
Krebber
H.
,
Kempf
T.
,
Hermes
I.
,
Ponstingl
H.
(
1995
).
Human RanGTPase-activating protein RanGAP1 is a homologue of yeast Rna1p involved in mRNA processing and transport.
Proc. Nat. Acad. Sci. USA
92
,
1749
1753
Boguski
M. S.
,
McCormick
F.
(
1993
).
Proteins regulating Ras and its relatives.
Nature
366
,
643
653
Bun-ya
M.
,
Harashima
S.
,
Oshima
Y.
(
1992
).
Putative GTP-binding protein, Gtr1, associated with the function of the Pho84 inorganic phosphate transporter in Saccharomyces cerevisiae.
Mol. Cell. Biol
12
,
2958
2966
Chuang
R.-Y.
,
Weaver
P. L.
,
Liu
Z.
,
Chang
T.-H.
(
1997
).
Requirement of the DEAD-Box protein Ded1p for messenger RNA translation.
Science
275
,
1468
1471
Clark
K. L.
,
Sprague
G. F.
Jr.
(
1989
).
Yeast pheromone response pathway: characterization of a suppressor that restores mating to receptorless mutants.
Mol. Cell. Biol
9
,
2682
2694
Dasso
M.
,
Seki
T.
,
Azuma
Y.
,
Ohba
T.
,
Nishimoto
T.
(
1994
).
A mutant form of the Ran/TC4 protein disrupts nuclear function in Xenopus laevis egg extracts by inhibiting the RCC1 protein, a regulator of chromosome condensation.
EMBO J
13
,
5732
5744
Drivas
G. T.
,
Shih
A.
,
Coutavas
E.
,
Rush
M. G.
,
D'Eustachio
P.
(
1990
).
Characterization of four novel RAS-related genes expressed in a human teratocarcinoma cell line.
Mol. Cell. Biol
10
,
1793
1798
Fu
X.
,
Maniatis
T.
(
1990
).
Factor required for mammalian splicesome assembly is localized to discrete regions in the nucleus.
Nature
343
,
437
441
Guan
K.
,
Dixon
J. E.
(
1991
).
Eukaryotic proteins expressed in Escherichia coli: an improved thrombin cleavage and purification procedure of fusion proteins with glutathione S-transferase.
Anal. Biochem
192
,
262
267
Gluzman
Y.
(
1981
).
SV40-transformed simian cells support the replication of early SV40 mutant.
Cell
23
,
175
182
Hopp
T. P.
,
Prickett
K. S.
,
Price
V. L.
,
Libby
R. T.
,
March
C. J.
,
Cerretti
D. P.
,
Urdal
D. L.
,
Conlon
P. J.
(
1988
).
A short polypeptide marker sequence useful for recombinant protein identification and purification.
Biotechnology
6
,
1204
1210
Hopper
A. K.
,
Banks
F.
,
Evangelidis
V.
(
1978
).
A yeast mutant which accumulates precursor tRNA.
Cell
19
,
211
219
Hutchison
H. T.
,
Hartwell
L. H.
,
McLaughlin
C. S.
(
1969
).
Temperature-sensitive yeast mutant defective in ribonucleic acid production.
J. Bacteriol
99
,
807
814
Ishov
A. M.
,
Stenberg
R. M.
,
Maul
G. G.
(
1997
).
Human cytomegalovirus immediate early interaction with host nuclear structures: definition of an immediate transcript environment.
J. Cell Biol
138
,
5
16
Kadowaki
T.
,
Goldfarb
D.
,
Spitz
L. M.
,
Tartakoff
A. M.
,
Ohno
M.
(
1993
).
Regulation of RNA processing and transport by a nuclear guanine nucleotide release protein and members of the Ras superfamily.
EMBO J
12
,
2929
2937
Lavau
C.
,
Marchio
A.
,
Fagioli
M.
,
Jansen
J.
,
Falini
B.
,
Lebon
P.
,
Grosveld
F.
,
Pandolfi
P. P.
,
Pelicci
P. G.
,
Dejean
A.
(
1995
).
The acute promyelocytic leukaemia-associated PML gene is induced by interferon.
Oncogene
11
,
871
876
Lee
A.
,
Clark
K. L.
,
Fleischmann
M.
,
Aebi
M.
,
Clark
M. W.
(
1994
).
Site-directed mutagenesis of the yeast PRP20/SRM1 gene reveals distinct activity domains in the protein product.
Mol. Gen. Genet
245
,
32
44
Matsumoto
T.
,
Beach
D.
(
1991
).
Premature initiation of mitosis in yeast lacking RCC1 or an interacting GTPase.
Cell
66
,
347
360
Maul
G. G.
,
Ishov
A. M.
,
Everett
R. D.
(
1996
).
Nuclear domain 10 as preexisting potential replication start sites of herpes simplex virus type-1.
Virology
217
,
67
75
Michael
S. F.
(
1994
).
Mutagenesis by incorporation of a phosphorylated oligo during PCR amplification.
BioTechniques
16
,
410
–.
Nakashima
N.
,
Hayashi
N.
,
Noguchi
E.
,
Nishimoto
T.
(
1996
).
Putative GTPase Gtr1p genetically interacts with the RanGTPase cycle in Saccharomyces cerevisiae.
J. Cell Sci
109
,
2311
2318
Nakashima
T.
,
Sekiguchi
T.
,
Kuraoka
A.
,
Fukushima
K.
,
Shibata
Y.
,
Komiyama
S.
,
Nishimoto
T.
(
1993
).
Molecular cloning of a human cDNA encoding a novel protein, DAD1 whose defect causes apoptotic cell death in hamster BHK21 cells.
Mol. Cell. Biol
13
,
6367
6374
Nishimoto
T.
,
Eilen
E.
,
Basilico
C.
(
1978
).
Premature chromosome condensation in a ts DNA-mutant of BHK cells.
Cell
15
,
475
483
Ninomiya-Tsuji
J.
,
Nomoto
S.
,
Yasuda
H.
,
Reed
S. I.
,
Matsumoto
K.
(
1991
).
Cloning of a human cDNA encoding a CDC2-related kinase by complementation of a budding yeast cdc28 mutation.
Proc. Nat. Acad. Sci. USA
88
,
9006
9010
Noguchi
E.
,
Hayashi
N.
,
Nakashima
N.
,
Nishimoto
T.
(
1997
).
Yrb2p,Nup2p-related yeast protein has functional overlap with Rna1p, yeast RanGAP protein.
Mol. Cell Biol
17
,
2235
2246
Ohtsubo
M.
,
Kai
R.
,
Furuno
N.
,
Sekiguchi
T.
,
Sekiguchi
M.
,
Hayashida
H.
,
Kuma
K.
,
Miyata
T.
,
Fukushige
S.
,
Murotu
T.
,
Matsubara
K.
,
Nishimoto
T.
(
1987
).
Isolation and characterization of the active cDNA of the human cell cycle gene (RCC1) involved in the regulation of onset of chromosome condensation.
Genes Dev
1
,
585
593
Pinol-Roma
S.
,
Dreyfuss
G.
(
1992
).
Shuttling of pre-mRNA binding proteins between nucleus and cytoplasm.
Nature
355
,
730
732
Ren
M.
,
Drivas
G.
,
D'Eustachio
P.
,
Rush
M. G.
(
1993
).
Ran/TC4: a small nuclear GTP-binding protein that regulates DNA synthesis.
J. Cell Biol
120
,
313
323
Sazer
S.
,
Nurse
P.
(
1994
).
A fission yeast RCC1-related protein is required for the mitosis to interphase transition.
EMBO J
13
,
606
615
Seki
T.
,
Hayashi
N.
,
Nishimoto
T.
(
1996
).
RCC1 in the Ran pathway.
J. Biochem
120
,
207
214
Schmid
S. R.
,
Linder
P.
(
1992
).
D-E-A-D protein family of putative RNA helicases.
Mol. Microbiol
6
,
283
292
Schurmann
A.
,
Brauers
A.
,
Maβmann
S.
,
Becker
W.
,
Joost
H.-G.
(
1995
).
Cloning of a novel family of mammalian GTP-binding proteins (RagA, RagBs, RagB1) with remote similarity to the Ras-related GTPase.
J. Biol. Chem
270
,
28982
28988
Weiss
K.
,
Rambaud
S.
,
Lavau
C.
,
Jansen
J.
,
Carvalho
T.
,
Carmo-Fonseca
M.
,
Lamond
A.
,
Dejean
A.
(
1994
).
Retinoic acid regulates aberrant nuclear localization of PML-RAR a in acute promyelocytic leukemia cells.
Cell
76
,
345
356
Xing
Y.
,
Johnson
C. V.
,
Moen
P. T.
Jr.
,
McNeil
J. A.
,
Lawrence
J. B.
(
1993
).
Nonrandom gene organization: Structural arrangements of specific pre-mRNA transcription and splicing with SC-35 domains.
J. Cell Biol
131
,
1635
1647
Yano
R.
,
Oakes
M. L.
,
Tabb
M. M.
,
Nomura
M.
(
1994
).
Yeast Srp1p has homology to armadillo/plakoglobin/-catenin and participates in apparently multiple nuclear functions including the maintenance of the nucleolar structure.
Proc. Nat. Acad. Sci. USA
91
,
6880
6884
This content is only available via PDF.