We have cloned the gene, vps34+, from the fission yeast Schizosaccharomyces pombe which encodes an 801 amino acid protein with phosphatidylinositol 3-kinase activity. The S. pombe Vps34 protein shares 43% amino acid sequence identity with the Saccharomyces cerevisiae Vps34 protein and 28% identity with the p110 catalytic subunit of the mammalian phosphatidylinositol 3-kinase. When the vps34+ gene is disrupted, S.pombe strains are temperature-sensitive for growth and the mutant cells contain enlarged vacuoles. Furthermore, while wild-type strains exhibit substantial levels of phosphatidylinositol 3-kinase activity, this activity is not detected in the vps34 delta strain. S.pombe Vps34p-specific antiserum detects a single protein in cells of -90 kDa that fractionates almost exclusively with the crude membrane fraction. Phosphatidylinositol 3-kinase activity also is localized mainly in the membrane fraction of wild-type cells. Immunoisolated Vps34p specifically phosphorylates phosphatidylinositol on the D-3 position of the inositol ring to yield phosphatidylinositol(3)phosphate. but does not utilize phosphatidylinositol(4)phosphate or phosphatidylinositol(4,5)bisphosphate as substrates. In addition, when compared to the mammalian p110 phosphatidylinositol 3-kinase, S. pombe Vps34p is relatively insensitive to the inhibitors wortmannin and LY294002. Together, these results indicate that S. pombe Vps34 is more similar to the phosphatidylinositol-specific 3-kinase, Vps34p from S. cerevisiae, and is distinct from the p110/p85 and G protein-coupled phosphatidylinositol 3-kinases from mammalian cells. These data are discussed in relation to the possible role of Vps34p in vesicle-mediated protein sorting to the S. pombe vacuole.

REFERENCES

Auger
K. R.
,
Serunian
L. A.
,
Soltoff
S. P.
,
Libby
P.
,
Cantley
L. C.
(
1989
).
PDGF-dependent tyrosine phosphorylation stimulates production of novel polyphosphoinositides in intact cells.
Cell
57
,
167
175
Auger
K.
,
Carpenter
C.
,
Schoelson
S.
,
Piwnica-Worms
H.
,
Cantley
L.
(
1992
).
Reconstitution of purified PtdIns 3-kinase with middle t/pp60c-src expressed in insect cells.
J. Biol. Chem
267
,
5408
5415
Bankaitis
V. A.
,
Johnson
L. M.
,
Emr
S. D.
(
1986
).
Isolation of yeast mutants defective in protein targeting to the vacuole.
Proc. Nat. Acad. Sci. USA
83
,
9075
9079
Cantley
L. C.
,
Auger
K. R.
,
Carpenter
C.
,
Duckworth
B.
,
Graziani
A.
,
Kapeller
R.
,
Soltoff
S.
(
1991
).
Oncogenes and signal transduction.
Cell
64
,
281
302
Carpenter
C. L.
,
Duckworth
B. C.
,
Auger
K. R.
,
Cohen
B.
,
Schaffhausen
B. S.
,
Cantley
L. C.
(
1990
).
Purification and characterization of phosphoinositide 3-kinase from rat liver.
J. Biol. Chem
265
,
19704
19711
Carpenter
C. L.
,
Auger
K. R.
,
Chanudhuri
M.
,
Yoakim
M.
,
Schaffhausen
B.
,
Shoelson
S.
,
Cantley
L. C.
(
1993
).
Phosphoinositide 3-kinase is activated by phosphopeptides that bind to the SH2 domains of the 85-kDa subunit.
J. Biol. Chem.
268
,
9478
9483
Christianson
T. W.
,
Sikorski
R. S.
,
Dante
M.
,
Shero
J. H.
,
Hieter
P.
(
1992
).
Multifunctional yeast high-copy-number shuttle vectors.
Gene
110
,
119
122
Dhand
R.
,
Hara
K.
,
Hiles
I.
,
Bax
B.
,
Gout
I.
,
Panayotou
G.
,
Fry
M. J.
,
Yonezawa
K.
,
Kasuga
M.
,
Waterfield
M. D.
(
1994
).
PI 3-kinase: structural and functional analysis of intersubunit interactions.
EMBO J
13
,
511
521
Dieckman
C. L.
,
Tzagoloff
A.
(
1985
).
Assembly of the mitochondrial membrane system. CBD6, a yeast nuclear gene necessary for synthesis of cytochrome b.
J. Biol. Chem
260
,
1513
1520
Feinberg
A. P.
,
Vogelstein
B.
(
1983
).
A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity.
Anal. Biochem
132
,
6
13
Flanagan
C. A.
,
Schnieders
E. A.
,
Emerick
A. W. R. K.
,
Admon
A.
,
Thorner
J.
(
1993
).
Phosphatidylinositol 4-kinase: gene structure and requirement for yeast cell viability.
Science
262
,
1444
1448
Gout
I.
,
Dhand
R.
,
Hiles
I. D.
,
Fry
M. J.
,
Panayotou
G.
,
Das
P.
,
Truong
O.
,
Totty
N. F.
,
Hsuan
J.
,
Booker
G. W.
,
Campbell
I. D.
,
Waterfield
M. D.
(
1993
).
The GTPase dynamin binds to and is activated by a subset of SH3 domains.
Cell
75
,
25
36
Grimm
C.
,
Kohli
J.
,
Murray
J.
,
Maundrell
K.
(
1988
).
Genetic engineering of Schizosaccharomyces: a system for gene disruption and replacement using the ura4 gene as a selectable marker.
Mol. Gen. Genet
215
,
81
86
Hawkins
P. T. J. T. R.
,
Stephens
L. R.
(
1992
).
Platelet-derived growth factor stimulates synthesis of PtdIns(3, 4, 5)-P3 by activating a PtdIns(4, 5)-P2 3-OH kinase.
Nature
358
,
157
159
Herman
P. K.
,
Emr
S. D.
(
1990
).
Characterization of VPS34, a gene required for vacuolar protein sorting and vacuole segregation in Saccharomyces cerevisiae.
Mol. Cell. Biol
10
,
6742
6754
Hiles
I. D.
,
Otsu
M.
,
Volinia
S.
,
Fry
M. J.
,
Guot
I.
,
Dhand
R.
,
Panayotou
G.
,
Ruiz-Larrea
F.
,
Thompson
A.
,
Totty
N. F.
,
Hsuan
J. J.
,
Courtneidge
S. A.
,
Parker
P. J.
,
Waterfield
M. D.
(
1992
).
Phosphatidylinositol 3-kinase: Structure and expression of the 110 kd catalytic subunit.
Cell
70
,
419
429
Hong
Z.
,
Verma
D. P. S.
(
1994
).
A phosphatidylinositol 3-kinase is induced during soybean nodule organogenesis and is associated with membrane proliferation.
Proc. Nat. Acad. Sci. USA
91
,
9617
9621
Hu
P.
,
Mondino
A.
,
Skolnik
E. Y.
,
Schlessinger
J.
(
1993
).
Cloning of a novel, ubiquitously expressed human phosphatidylinositol 3-kinase and identification of its binding site on p85.
Mol. Cell. Biol
13
,
7677
7688
Hu
P.
,
Schlessinger
J.
(
1994
).
Direct association of p110 beta phosphatidylinositol 3-kinase with p85 is mediated by an N-terminal fragment of p110 beta.
Mol. Cell. Biol
14
,
2577
2583
Joly
M.
,
Kazlauskas
A.
,
Fay
F. S.
,
Corvera
S.
(
1994
).
Disruption of PDGF receptor trafficking by mutation of its PI-3 kinase binding sites.
Science
263
,
684
687
Jones
E. W.
(
1977
).
Proteinase mutants of Saccharomyces cerevisiae.
Genetics
85
,
23
33
Kapeller
R.
,
Chakrabarti
R.
,
Cantley
L.
,
Fay
F.
,
Corvera
S.
(
1993
).
Internalization of activated platelet-derived growth factor receptor-phosphatidylinositol-3′ kinase complexes: potential interactions with the microtubule cytoskeleton.
Mol. Cell. Biol
13
,
6052
6063
Kleid
D. G.
,
Yansura
D.
,
Small
B.
,
Dowbenko
D.
,
Moore
M.
,
Robertson
B. H.
,
Bachrach
H. L.
(
1981
).
Cloned viral protein vaccine for foot-and-mouth disease: responses in cattle and swine.
Science
214
,
1125
1129
Klionsky
D. J.
,
Banta
L. M.
,
Emr
S. D.
(
1988
).
Intracellular sorting and processing of a yeast vacuolar hydrolase: Proteinase A propeptide contains vacuolar targeting information.
Mol. Cell. Biol
8
,
2105
2116
Klionsky
D. J.
,
Herman
P. K.
,
Emr
S. D.
(
1990
).
The fungal vacuole: Composition, function and biogenesis.
Microbiol. Rev.
54
,
266
292
Knighton
D. R.
,
Zheng
J. H.
,
Ten Eyck
L. F.
,
Ashford
V. A.
,
Xuong
N. H.
,
Taylor
S. S.
,
Sowadski
J. M.
(
1991
).
Crystal structure of the catalytic subunit of cyclic adenosine monophosphate-dependent protein kinase.
Science
253
,
407
414
Lam
K.
,
Carpenter
C. L.
,
Ruderman
N. B.
,
Friel
J. C.
,
Kelly
K. L.
(
1994
).
The phosphatidylinositol 3-kinase serine kinase phosphorylates IRS-1.
J. Biol. Chem
269
,
20648
20652
McLeod
M.
,
Stein
M.
,
Beach
D.
(
1987
).
The product of the mei3+ gene, expressed under control of the mating-type locus, induces meiosis and sporulation in fission yeast.
EMBO J
6
,
729
736
Moreno
S.
,
Klar
A.
,
Nurse
P.
(
1991
).
Molecular genetic analysis of fission yeast Schizosaccharomyces pombe.
Meth. Enzymol
194
,
795
823
Okada
T.
,
Kawano
Y.
,
Sakakibara
T.
,
Hazeki
O.
,
Ui
M.
(
1994
).
Essential role of phosphatidylinositol 3-kinase in insulin-induced glucose transport and antilipolysis in rat adipocytes- Studies with a selective inhibitor wortmannin.
J. Biol. Chem
269
,
3568
3573
Okada
T.
,
Sakuma
L.
,
Fukui
Y.
,
Hazeki
O.
,
Ui
M.
(
1994
).
Blockage of chemotactic peptide-induced stimulation of neutrophils by wortmannin as a result of selective inhibition of phosphatidylinositol 3-kinase.
J. Biol. Chem
269
,
3563
3567
Okazaki
K.
,
Okazaki
K.
,
Kume
K.
,
Jinno
S.
,
Tanaka
K.
,
Okayama
H.
(
1990
).
High-frequency transformation method and library transducing vectors for cloning mammalian cDNAs by trans-complementation of Schizosaccharomyces pombe.
Nucl. Acids Res
18
,
6485
6489
Otsu
M.
,
Hiles
I.
,
Gout
I.
,
Fry
M. J.
,
Ruiz-Larrea
F.
,
Panayotou
G.
,
Thompson
A.
,
Dhand
R.
,
Hsuan
J.
,
Totty
N.
,
Smith
A. D.
,
Morgan
S. J.
,
Courtneidge
S. A.
,
Parker
P. J.
,
Waterfield
M. D.
(
1991
).
Characterization of two 85 kd proteins that associate with receptor tyrosine kinases, middle-T/pp60 c-src complexes, and PI3-kinase.
Cell
65
,
91
104
Panayotou
G.
,
Bax
B.
,
Gout
I.
,
Federwisch
M.
,
Wroblowski
B.
,
Dhand
R.
,
Fry
M. J.
,
Blundell
T. L.
,
Wollmer
A.
,
Waterfield
M. D.
(
1992
).
Interaction of the p85-subunit of Pl 3-kinase and its N-terminal SH2-domain with a PDGF receptor phosphorylation site- structural features and analysis of conformational changes.
EMBO J
11
,
4261
4272
Robinson
J. S.
,
Klionsky
D. J.
,
Banta
L. M.
,
Emr
S. D.
(
1988
).
Protein sorting in Saccharomyces cerevisiae: isolation of mutants defective in the delivery and processing of multiple vacuolar hydrolases.
Mol. Cell. Biol
8
,
4936
4948
Rothman
J. H.
,
Stevens
T. H.
(
1986
).
Protein sorting in yeast: Mutants defective in vacuole biogenesis mislocalization vacuolar proteins into the late secretory pathway.
Cell
47
,
1041
1051
Rothman
J. H.
,
Yamashiro
C. T.
,
Kane
P. M.
,
Stevens
T. H.
(
1989
).
Protein targeting to the yeast vacuole.
Trends Biochem. Sci
14
,
347
350
Saiki
R. K.
,
Gelfand
D. H.
,
Stoffel
S.
,
Scharf
S. J.
,
Higuchi
R.
,
Horn
G. T.
,
Mullis
K. B.
,
Erlich
H. A.
(
1988
).
Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase.
Science
239
,
88
91
Schu
P. V.
,
Takegawa
K.
,
Fry
M. J.
,
Stack
J. H.
,
Waterfield
M. D.
,
Emr
S. D.
(
1993
).
Phosphatidylinositol 3-kinase encoded by yeast VPS34 gene essential for protein sorting.
Science
260
,
88
91
Segal
A. W.
,
Abo
A.
(
1993
).
The biochemical basis of the NADPH oxidase of phagocytes.
Trends Biochem. Sci
18
,
43
47
Stack
J. H.
,
Herman
P. K.
,
Schu
P. V.
,
Emr
S. D.
(
1993
).
A membrane-associated complex containing the Vps15 protein kinase and the Vps34 PI 3-kinase is essential for protein sorting to the yeast lysosome-like vacuole.
EMBO J
12
,
2195
2204
Stack
J. H.
,
Emr
S. D.
(
1994
).
Vps34p required for yeast vacuolar protein sorting is a multiple specificity kinase that exhibits both protein kinase and phosphatidylinositol-specific PI 3-kinase activities.
J. Biol. Chem
269
,
31552
31562
Stack
J. H.
,
DeWald
D. B.
,
Takegawa
K.
,
Emr
S. D.
(
1995
).
Regulatory interactions between the Vps15 protein kinase and the Vps34 PI 3-kinase essential for signaling vesicle-mediated protein transport to the vacuole in yeast.
J. Cell Biol
129
,
321
334
Stephens
L.
,
Hughes
K.
,
Irvine
R. F.
(
1991
).
Pathways of phosphatidylinositol(3,4,5)-trisphosphate synthesis in activated neutrophils.
Nature
351
,
33
39
Stephens
L. R.
,
Jackson
T. R.
,
Hawkins
P. T.
(
1993
).
Agonist-stimulated synthesis of phosphatidylinositol(3,4,5)-trisphosphate: a new intracellular signalling system.
Biochim. Biophys. Acta
1179
,
27
75
Stephens
L.
,
Cooke
F. T.
,
Walters
R.
,
Jackson
T.
,
Volina
S.
,
Gout
I.
,
Waterfield
M. D.
,
Hawkins
P. T.
(
1994
).
Characterization of a phosphatidylinositol-specific phosphoinositide 3-kinase from mammalian cells.
Curr. Biol
4
,
203
214
Stephens
L.
,
Smrcka
A.
,
Cooke
F.
,
Jackson
T. R.
,
Sternweis
P. C.
,
Hawkins
P. T.
(
1994
).
A novel phosphoinositide 3 kinase activity in myeloid-derived cells is activated by G protein beta gamma subunits.
Cell
77
,
83
93
Thelen
M.
,
Wymann
M. P.
,
Langen
H.
(
1994
).
Wortmannin binds specifically to 1-phosphatidylinositol 3-kinase while inhibiting guaninenucleotide-binding protein-coupled receptor signaling in neutrophil leukocytes.
Proc. Nat. Acad. Sci. USA
91
,
4960
4964
Vlahos
C. J.
,
Matter
W. F.
,
Hui
K. Y.
,
Brown
R. F.
(
1994
).
A specific inhibitor of phosphatidylinositol 3-kinase, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002).
J. Biol. Chem
269
,
5241
5248
Volinia
S.
,
Dhand
R.
,
Vanhaesebroeck
B.
,
MacDougall
L. K.
,
Stein
R.
,
Zvelebil
M. J.
,
Domin
J.
,
Panaretou
C.
,
Waterfield
M. D.
(
1995
).
A human phosphatidylinositol 3-kinase complex related to the yeast Vps34p-Vps15p protein sorting system.
EMBO J
14
,
3339
3348
Walsh
J. P.
,
Caldwell
K. K.
,
Majerus
P. W.
(
1991
).
Formation of phosphatidylinositol 3-phosphate by isomerization from phosphatidylinositol 4-phosphate.
Proc. Nat. Acad. Sci. USA
88
,
9184
9187
Welters
P.
,
Takegawa
K.
,
Emr
S. D.
,
Chrispeels
M. J.
(
1994
).
AtVPS34, a phosphatidylinositol 3-kinase of Arabidopsis thaliana is an essential protein with homology to a calcium-dependent lipid binding domain.
Proc. Nat. Acad. Sci. USA
91
,
11398
11402
Wennström
S.
,
Hawkins
P.
,
Cooke
F.
,
Hara
K.
,
Yonezawa
K.
,
Kasuga
M.
,
Jackson
T.
,
Claesson-Welsh
L.
,
Stephens
L.
(
1994
).
Activationof phosphoinositide 3-kinase is required for PDGF-stimulated membrane ruffling.
Curr. Biol
4
,
385
393
Yano
H.
,
Nakanishi
S.
,
Kimura
K.
,
Hanai
N.
,
Saitoh
Y.
,
Fukui
Y.
,
Nonomura
Y.
,
Matsuda
Y.
(
1993
).
Inhibition of histamine secretion by wortmannin through the blockade of phosphatidylinositol 3-kinase in RBL-2H3 cells.
J. Biol. Chem
268
,
25846
25856
Yoshida
S.
,
Ohya
Y.
,
Goebl
M.
,
Nakano
A.
,
Anraku
Y.
(
1994
).
A novel gene, STT4, encodes a phosphatidylinositol 4-kinase in the PKC1 protein kinase pathway of Saccharomyces cerevisiae.
J. Biol. Chem
269
,
1166
1172
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