1 Hans-Knöll-Institute for Natural Products Research, Department of Infection Biology, Beutenbergstrasse 11, D-07745 Jena, Germany
2 University of Applied Sciences, Tatzendpromenade 1b, D-07745 Jena, Germany
Correspondence
Raimund Eck
reck{at}pmail.hki-jena.de
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In C. albicans, the phosphatidylinositol (PI) 3-kinase Vps34p is involved in virulence. The C. albicans vps34 null mutant is unable to form hyphae on various solid media, shows a significantly delayed yeast to hyphae transition in liquid media, is hypersensitive to high temperature and hyperosmotic stress and exhibits reduced adherence to human cells. In addition, the vps34 null mutant displays enlarged and electron-transparent vacuoles and is avirulent in a mouse model of systemic candidiasis (Eck et al., 2000; Bruckmann et al., 2000
; 2001
).
PI 3-kinases control a wide variety of cellular processes in eukaryotic cells, including mitogenesis, protection from apoptosis, growth factor receptor down-regulation, stimulation of glucose uptake, endocytosis, actin cytoskeleton rearrangement and intracellular protein/membrane trafficking (DeCamilli et al., 1996; Toker & Cantley, 1997
). PI 3-kinases are subdivided into three classes, based on sequence similarities, substrate specificity and regulatory properties (Vanhaesebroeck et al., 1997
). Class I PI 3-kinases represent dual-specificity enzymes, and display both lipid kinase and protein kinase activity. The generation of a lipid-kinase-defective human PI 3-kinase
with a defective putative PI-binding domain was used to show the transphosphorylation of the PI 3-kinase adapter protein p101 and the MAP protein kinase MEK-1 in vitro (Stoyanov et al., 1995
; Bondeva et al., 1998
; Bondev et al., 1999
).
The class III PI 3-kinase Vps34p (Vps: vacuolar protein sorting) represents the only PI 3-kinase activity in the yeast Saccharomyces cerevisiae (Schu et al., 1993; Vanhaesebroeck et al., 1997
). ScVps34p regulates intracellular protein trafficking to the vacuole, retrograde endosome-to-Golgi transport, autophagocytosis and vacuole acidification (Herman & Emr, 1990
; Zhou et al., 1995
; Burda et al., 2002
; Kihara et al., 2001
; Munn & Riezman, 1994
). Two distinct Vps34p-containing protein complexes were identified in S. cerevisiae. (Kihara et al., 2001
). ScVps34p shows lipid kinase and autophosphorylation activity, and the protein is predominantly phosphorylated on serine residues (Stack & Emr, 1994
). ScVps34 proteins containing mutated residues in the putative lipid kinase domain (amino acid positions 731, 735, 736 and 749) are defective in both autophosphorylation and lipid kinase function (Stack & Emr, 1994
; Schu et al., 1993
).
In this study, we generated a Vps34T mutant protein of C. albicans with specifically inactivated lipid kinase activity. A Candida mutant strain containing the lipid-kinase-defective gene was generated. The phenotype of this strain indicated that the lipid kinase activity is essential for the virulence, dimorphism, adhesion, vesicular protein transport and vacuole morphology of C. albicans. Additional activities of Vps34p, which are mainly involved in the stress response, were shown in vivo.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
Enzyme assays.
PI 3-kinase assays were carried out essentially as described by Schu et al. (1993). Approximately 0·15 µg of recombinant protein was assayed in 50 µl reactions containing 20 mM HEPES, pH 7·5, 10 mM MgCl2, 0·2 mg sonicated phosphatidylinositol ml1, 60 µM ATP and 0·1 µCi [
-32P]ATP ml1. The mixtures were incubated at 30 °C for 15 min, reactions were terminated by the addition of 80 µl 1 M HCl and lipids were extracted with 160 µl chloroform/methanol (1 : 1). The organic phase was dried, samples were resuspended in chloroform and spotted onto Silica gel 60 TLC plates (Merck), and the plates were developed in a borate buffer system (Walsh et al., 1991
). Labelled phosphoinositides were analysed using a phosphoimager.
Autophosphorylation assays were carried out as described by Czupalla et al. (2003). Recombinant protein (200 ng) was assayed in 50 µl reactions containing 0·1 % BSA, 1 mM EGTA, 0·2 mM EDTA, 7 mM MgCl2, 10 mM MnCl2, 100 mM NaCl, 40 mM HEPES, pH 7·4, 1 mM DTT, 1 mM
-glycerophosphate, 25 µM ATP and 0·1 µCi [
-32P]ATP ml1. The reaction was stopped after incubation at 30 °C for 30 min by adding 17 µl 4x sample buffer according to Laemmli (1970)
. The proteins were separated on SDS-polyacrylamide gels, dried and autoradiographic signals were analysed using a phosphoimager.
Integration of the chimeric VPS34TOR gene in C. albicans.
The chimeric VPS34TOR gene was introduced into the vps34 null mutant strain CAV4 (ura) using the 6·0 kb BbvI/HindIII insert of pKEUT (Fig. 2). This insert contains the chimeric VPS34TOR gene, and includes upstream and downstream sequences to allow homologous recombination, as well as the URA3 gene as a selectable marker. Transformations were performed by electroporation, as described by DeBacker et al. (1999)
. Chromosomal DNA isolated from selected clones was digested with HindIII/KpnI and analysed by Southern hybridization with an [
-32P]dCTP-labelled EcoRI/HindIII 4·9 kb insert of plasmid pKE2 (Bruckmann et al., 2000
).
|
Adherence assay.
The ability of C. albicans strains SC5314, CAV3 and CAV9 to adhere to human buccal epithelial cells (HBEC) in vitro were examined by a visual assay (Bailey et al., 1995). The Candida strains were grown, washed and counted according to the method of Bailey et al. (1995)
. HBEC from a male volunteer were washed with 1x PBS (Invitrogen) and counted. A total of 107 Candida cells were incubated with 105 HBEC in 1x PBS at 37 °C for 2 h. The cells were then transferred to microscope slides. Adherence was expressed as the percentage of HBEC with adhering Candida cells.
Virulence studies.
Male outbred NMRI mice (Harlan-Winkelmann), 6 weeks old, were housed five per cage and checked daily. C. albicans cells were grown in Sabouraud dextrose broth at 28 °C until late-exponential phase. Cells were washed three times and resuspended in 0·9 % NaCl. Portions (200 µl) of suspensions containing 5x106, 5x105 and 5x104 cells were used to infect immunocompetent mice by intravenous injection into the lateral tail vein. Survival was monitored for 21 days. To quantify kidney colonization by C. albicans, mice were sacrificed 3 days and 21 days after injection, and kidneys were homogenized in 3 ml physiological NaCl buffer. Serially diluted suspensions were then plated on YPD agar. After 3 days growth at 28 °C, numbers of Candida colonies were counted. Homogenized kidney material was also fixed with 10 % formaldehyde and stained with 25 mg Calcofluor White ml1 to detect C. albicans cells.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
It was necessary to determine the lipid kinase activity of chimeric Vps34Tp, and therefore the formation of PI 3-phosphate (PI 3-P) by Vps34Tp was tested. Phosphorylated lipids were separated by TLC and monitored using a phosphoimager. The chimeric Vps34T protein did not form PI 3-P in vitro, in contrast to wild-type Vps34p (Fig. 1c).
The autophosphorylation activity of chimeric Vps34Tp was tested by assaying the incorporation of radioactive phosphate. Both Vps34Tp and the wild-type Vps34 protein incorporate 32P, and thus both proteins are able to autophosphorylate in vitro (Fig. 1b). These results prove that the exchange of six amino acids within the putative PI-binding domain of Vps34p specifically inactivates the lipid kinase, but preserves the autophosphorylation activity.
Generating a lipidkinase-defective Vps34p-containing C. albicans mutant strain
In order to investigate the role of the lipid kinase activity of Vps34p and that of the other activities, such as autophosphorylation, separately in vivo, a C. albicans mutant strain containing the chimeric VPS34TOR gene lacking a functional lipid kinase domain was generated. To this end, a 6·0 kb BbvI/HindIII fragment of pKEU1T containing the chimeric VPS34TOR gene and the marker gene URA3 was transformed to C. albicans vps34 ura null mutant strain CAV4 (Fig. 2a). Southern analysis of HindIII/KpnI-digested chromosomal DNA proved the proper integration of the genes. The 7·0 kb fragment in lane 1 represents the VPS34 gene of C. albicans wild-type strain SC5314 (Fig. 2b
). The loss of this 7·0 kb fragment and the appearance of 2·5 and 2·7 kb fragments (Fig. 2b
, lane 2) is consistent with the replacement of both VPS34 alleles by hisG in strain CAV4. Reintegration of VPS34 and URA3 into strain CAV4 is demonstrated by the occurrence of an additional 8·4 kb fragment in the heterozygous VPS34 revertant strain CAV5 (lane 3). In the Vps34p lipid-kinase-defective mutant strain CAV9, this fragment is restricted by an additional KpnI restriction site in the integrated chimeric VPS34TOR gene to form 3·1 and 5·3 kb fragments (lane 4). These results prove the correct integration of VPS34TOR.
The lipid kinase activity of Vps34p plays a role in the virulence of C. albicans
The growth of the lipid-kinase-defective mutant strain CAV9 was examined, as the growth rate is important for the virulence of C. albicans. Both the CAV9 and CAV3 mutant strains showed identical delayed growth. However, the growth rates of the mutants were similar to the wild-type strain SC5314. The delayed growth of the mutants may partly contribute to their avirulence. However, reduced growth is not always connected to avirulence (Augsten et al., 2002).
In order to identify whether the lipid kinase activity of Vps34p is involved in virulence or not, we tested the lipid-kinase-defective strain CAV9 in a mouse model for systemic candidiasis. We found that mutant strain CAV9 was non-virulent in mice. All mice infected with the mutant strain survived for three weeks: even animals infected with a high number of Candida cells (5x106). An identical survival rate has been shown for the vps34 null mutant strain CAV3 (Bruckmann et al., 2000). In contrast, all mice infected with the same cell number of the wild-type strain SC5314 died after 2 days, and 20 % of the mice survived for three weeks after infection with 5x104 cells. Nearly the same mortality rate has been observed for mice infected with the VPS34 heterozygous revertant strain CAV5 (Bruckmann et al., 2000
). The avirulence of both the lipid-kinase-defective Vps34p-containing mutant strain CAV9 and the vps34 null mutant strain CAV3 shows the central role of the lipid kinase in virulence (Fig. 3
).
|
The low c.f.u. of kidneys infected with the vps34 null mutant and the Vps34p lipid-kinase-defective mutant may indicate a lower ability of the mutants to adhere to endothelial cells in vivo, resulting in rapid clearance from the blood. Thus, the abilities of SC5314, CAV3 and CAV9 to adhere to HBEC in vitro were determined in a visual assay (Bailey et al., 1995). The proportion of HBEC with adhering wild-type Candida cells was 85 % (n=3). The proportion of HBEC with CAV3 or CAV9 cells on the surface was lower, at 45 and 50 %, respectively.
Lipid kinase activity of CaVps34p is required for hyphal growth
The switch between yeast and hyphae is considered important to the virulence of C. albicans. The Vps34p lipid-kinase-defective strain CAV9 was examined in liquid medium and on solid medium under different hyphae-inducing conditions in order to test the influence of Vps34p lipid kinase activity on hyphal growth.
First, hyphal growth was induced in liquid YPD medium supplemented with serum. Under these conditions, the mutant strain CAV9 showed delayed hyphal formation. Moreover, approximately 80 % of the hyphae were pseudohyphae. The delayed hyphal formation was more pronounced in the vps34 null mutant strain CAV3 (Fig. 4a). Identical results were obtained in liquid Spider medium (data not shown).
|
The differences in hyphal growth observed between the lipid-kinase-defective strain CAV9 and the wild-type strain indicate that the lipid kinase activity of Vps34p regulates the hyphal formation of C. albicans.
Lipid kinase activity of Vps34p is involved in transport of prevacuolar endocytic compartments to the vacuole and vacuole morphology
In the yeasts S. cerevisiae and C. albicans, Vps34p is involved in the transport of prevacuolar vesicles to the vacuole during the overlapping late steps of endocytosis and the CPY (carboxypeptidase Y) protein transport pathway from the late Golgi to the vacuole (Wurmser & Emr, 1998; Bruckmann et al., 2001
). This function may be analysed by following the distribution of the fluorescent lipophilic dye FM4-64. In yeast, this dye allows endocytotic uptake and vesicle-mediated transport to the vacuole to be monitored (Vida & Emr, 1995
). In order to investigate the influence of the lipid kinase activity of Vps34p in this process, the endocytic transport of the dye was analysed in the mutant strain CAV9, containing the lipid-kinase-defective Vps34T protein, and was compared to the distribution in the C. albicans wild-type strain and the vps34 null mutant strain CAV3 by fluorescence microscopy (Fig. 5
). The mutant strain CAV9 showed weak fluorescent staining of the vacuole membrane, but prevacuolar endocytic compartments in the cytoplasm were stained. In the wild-type strain SC5314, the dye had reached the vacuole, and a typical ring staining pattern was observed. This difference indicates that the lack of lipid kinase activity in the mutant strain CAV9 results in defects in the transport/fusion of prevacuolar endocytic vesicles to the vacuole. Thus, the lipid kinase activity of Vps34p seems necessary for a late step in endocytic protein transport to the vacuole.
|
Activities of Vps34p different from the lipid kinase function are involved in high-temperature and osmotic stress response
The C. albicans vps34 null mutant strain CAV3 is more sensitive to high temperature and hyperosmotic stress than the wild-type strain SC5314 (Bruckmann et al., 2000). Therefore, the contribution of the lipid kinase activity of Vps34p to high-temperature and osmotic stress responses was determined. The growth of the C. albicans Vps34p lipid-kinase-defective strain CAV9, the wild-type strain SC5314, the VPS34 revertant strain CAV5 and the vps34 null mutant strain CAV3 was assayed on YPD plates at 30, 38 and 40 °C. The osmotic sensitivity was examined on solid YPD medium containing NaCl or KCl at 1·2 M or 1·5 M. The mutant strain CAV9 showed a reduced stress resistance compared to the wild-type strain and the revertant strain CAV5. However, the vps34 null mutant strain CAV3 was clearly more sensitive to high-temperature and osmotic stress than the CAV9 strain (Fig. 6
). These findings suggest that an activity distinct from the lipid kinase activity of Vps34p plays a role in the stress response of C. albicans.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In general, manipulations of the lipid-binding domain of the PI 3-kinase Vps34 interfere with both lipid kinase and protein kinase activity (Wymann et al., 1996; Stack & Emr, 1994
). In human cells, a lipid-kinase-defective PI 3-kinase
with remaining protein kinase function was constructed, but little is known about the specific role of each of the two enzymic functions in vivo (Bondeva et al., 1998
).
Here, we describe the exchange of six amino acids from the putative PI-interacting domain of Vps34p with the homologous region of the PI 3-kinase-like Tor protein, resulting in the chimeric Vps34T protein. Probably, the exchange of the interaction domain blocks binding of the substrate PI to Vps34Tp, while autophosphorylation is not influenced. This is surprising, because the change of asparagine, localized eight amino acids upstream from the first amino acid of the exchanged region, causes the loss of autophosphorylation activity in Vps34p of S. cerevisiae (Stack & Emr, 1994).
The chimeric Vps34T protein lacking lipid kinase activity but retaining autophosphorylation activity allows the separate study of the role of the lipid kinase activity of the PI 3-kinase Vps34p. A C. albicans lipid-kinase-defective strain, CAV9, was generated, which has the chimeric Vps34TOR gene integrated. This gene encodes a protein which specifically lacks lipid kinase activity. Otherwise, it is identical to the wild-type Vps34p. Thus, the Vps34p lipid-kinase-defective strain CAV9 and the VPS34 revertant strain CAV5, which shows a phenotype nearly identical to that of the wild-type strain SC5314, differ in only six amino acids in the VPS34 gene; this causes the lack of Vps34Tp lipid kinase activity in the mutant strain CAV9, although the autophosphorylation activity is maintained. The differences in the phenotypes observed here between the mutant strain CAV9 and the CAV5 or wild-type strain indicate an important role for the lipid kinase activity of Vps34p, as most likely this activity was specifically eliminated. Therefore, the phenotypic differences suggest that lipid kinase activity plays a role in virulence, hyphal growth and adhesion, as well as in vacuole morphology and vesicular protein transport to the vacuoles. This conclusion is confirmed by several defective features of the vps34 null mutant strain CAV3 which are identical to the phenotypes observed for the mutant strain CAV9. These phenotypes are likely connected to the lipid kinase activity of Vps34p, because both strains lack this activity.
However, minor differences exist between the Vps34p lipid-kinase-defective strain CAV9 and the vps34 null mutant strain CAV3 regarding hyphal formation. The delay of hyphal development of the mutant strains CAV9 and CAV3 may be connected to delayed growth. However, the delay of hyphal growth is lower for CAV9 than for CAV3. In addition, although both mutant strains do not form hyphae on hyphae-inducing solid agar, their colony morphology differs clearly. As both CAV9 and CAV3 are avirulent in a mouse model of systemic candidiasis, the differences in hyphae growth and colony morphology do not seem to be important for virulence.
The different phenotypes observed between the Vps34p lipid-kinase-defective strain CAV9 and the vps34 null mutant strain CAV3 are indicative of additional activities for the Vps34 protein in vivo because the lipid kinase activity was absent in both CAV9 and CAV3. These phenotypes are connected to stress response.
The generation of Vps34Tp, which lacks lipid kinase but retains autophosphorylation activity, allowed the assay of the role of lipid kinase activity in vivo. Moreover, the functions of lipid-kinase-independent activities in the stress response were shown. These results indicate that the lipid kinase activity of Vps34p plays a role in virulence, and will help to identify the proper signalling pathways required for virulence.
![]() |
ACKNOWLEDGEMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Bailey, A., Wadsworth, E. & Calderone, R. (1995). Adherence of Candida albicans to human buccal epithelial cells: host-induced protein synthesis and signaling events. Infect Immun 63, 569572.[Abstract]
Bondev, A., Rubio, I. & Wetzker, R. (1999). Differential regulation of lipid and protein kinase activities of phosphoinositide 3-kinase gamma in vitro. Biol Chem 380, 13371340.[CrossRef][Medline]
Bondeva, T., Pirola, L., Bulgarelli-Leva, G., Rubio, I., Wetzker, R. & Wymann, M. P. (1998). Bifurcation of lipid and protein kinase signals of PI3Kgamma to the protein kinases PKB and MAPK. Science 282, 293296.
Bruckmann, A., Künkel, W., Härtl, A., Wetzker, R. & Eck, R. (2000). A phosphatidylinositol 3-kinase of Candida albicans influences adhesion, filamentous growth, and virulence. Microbiology 146, 27552764.[Medline]
Bruckmann, A., Künkel, W., Augsten, K., Wetzker, R. & Eck, R. (2001). The deletion of CaVPS34 in the human pathogenic yeast Candida albicans causes defects in vesicle-mediated protein sorting and nuclear segregation. Yeast 18, 343353.[CrossRef][Medline]
Burda, P., Padilla, S. M., Sarkar, S. & Emr, S. D. (2002). Retromer function in endosome-to-Golgi retrograde transport is regulated by the yeast Vps34 PtdIns 3-kinase. J Cell Sci 115, 38893900.
Cutler, J. E. (1991). Putative virulence factors of Candida albicans. Annu Rev Microbiol 45, 187218.[CrossRef][Medline]
Czupalla, C., Culo, M., Muller, E. C., Brock, C., Reusch, H. P., Spicher, B., Krause, E. & Nürnberg, B. (2003). Identification and characterization of the autophosphorylation sites of phosphoinositide 3-kinase isoforms beta and gamma. J Biol Chem 278, 1153611545.
DeBacker, M. D., Maes, D., Vandoninck, S., Logghe, M., Contreras, R. & Luyten, W. H. (1999). Transformation of Candida albicans by electroporation. Yeast 15, 16091618.[CrossRef][Medline]
DeCamilli, P., Emr, S. D., McPherson, P. S. & Novick, P. (1996). Phosphoinositides as regulators in membrane traffic. Science 271, 15331539.[Abstract]
Eck, R., Bruckmann, A., Wetzker, R. & Künkel, W. (2000). A phosphatidylinositol 3-kinase of Candida albicans: molecular cloning and characterization. Yeast 16, 933944.[CrossRef][Medline]
Fonzi, W. A. & Irwin, M. Y. (1993). Isogenic strain construction and gene mapping in Candida albicans. Genetics 134, 717728.
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, 67426754.[Medline]
Kihara, A., Noda, T., Ishihara, N. & Ohsumi, Y. (2001). Two distinct Vps34 phosphatidylinositol 3-kinase complexes function in autophagy and Carboxypeptidase Y sorting in Saccharomyces cerevisiae. J Cell Biol 152, 519530.
Köhler, J. R. & Fink, G. R. (1996). Candida albicans strains heterozygous and homozygous for mutations in mitogen-activated protein kinase signaling components have defects in hyphal development. Proc Natl Acad Sci U S A 93, 1322313228.
Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680685.[Medline]
Munn, A. L. & Riezman, H. (1994). Endocytosis is required for the growth of vacuolar H(+)-ATPase-defective yeast: identification of six new END genes. J Cell Biol 127, 373386.[Abstract]
Odds, F. C. (1994). Pathogenesis of Candida infections. J Am Acad Dermatol 31, S2S5.[Medline]
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, 8891.[Medline]
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, 3155231562.
Stoyanov, B., Volinia, S., Hanck, T. & 7 other authors (1995). Cloning and characterization of a G protein-activated human phosphoinositide-3 kinase. Science 269, 690693.[Medline]
Toker, A. & Cantley, L. C. (1997). Signalling through the lipid products of phosphoinositide-3-OH kinase. Nature 387, 673676.[CrossRef][Medline]
Vanhaesebroeck, B., Leevers, S. J., Panayotou, G. & Waterfield, M. D. (1997). Phosphoinositide 3-kinases: a conserved family of signal transducers. Trends Biochem Sci 22, 267272.[CrossRef][Medline]
Vida, T. A. & Emr, S. D. (1995). A new vital stain for visualizing vacuolar membrane dynamics and endocytosis in yeast. J Cell Biol 128, 779792.[Abstract]
Walsh, J. P., Caldwell, K. K. & Majerus, P. W. (1991). Formation of phosphatidylinositol 3-phosphate by isomerization from phosphatidylinositol 4-phosphate. Proc Natl Acad Sci U S A 88, 91849187.[Abstract]
Wurmser, A. E. & Emr, S. D. (1998). Phosphoinositide signaling and turnover: PtdIns(3)P, a regulator of membrane traffic, is transported to the vacuole and degraded by a process that requires lumenal vacuolar hydrolase activities. EMBO J 17, 49304942.
Wymann, M. P., Bulgarelli-Leva, G., Zvelebil, M. J., Pirola, L., Vanhaesebroeck, B., Waterfield, M. D. & Panayotou, G. (1996). Wortmannin inactivates phosphoinositide 3-kinase by covalent modification of Lys-802, a residue involved in the phosphate transfer reaction. Mol Cell Biol 16, 17221733.[Abstract]
Zhou, K., Takegawa, K., Emr, S. D. & Firtel, R. A. (1995). A phosphatidylinositol (PI) kinase gene family in Dictyostelium discoideum: biological roles of putative mammalian p110 and yeast Vps34p PI 3-kinase homologs during growth and development. Mol Cell Biol 15, 56455656.[Abstract]
Received 14 May 2004;
revised 30 July 2004;
accepted 14 September 2004.
HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
J MED MICROBIOL | ALL SGM JOURNALS |