bluuebunny/crossref_metadata_2025 huggingface dataset:
Molecular Analysis ofCPRα, aMATα-Specific Pheromone Receptor Gene ofCryptococcus neoformans
Authors: Chung Seyung , Karos Marvin , Chang Yun C. , Lukszo Jan , Wickes Brian L. , Kwon-Chung Kyung J.
Published: December 2002

The putativeCryptococcus neoformanspheromone receptor geneCPRαwas isolated and studied for its role in mating and filamentation.CPRαisMATαspecific and located adjacent toSTE12αat theMATαlocus. It encodes a protein which possesses high sequence similarity to the seven-transmembrane class of G-protein-coupled pheromone receptors reported for other basidiomycetous fungi. Strains containing a deletion of theCPRαgene exhibited drastic reductions in mating efficiency but were not completely sterile.Δcprα cells displayed wild-type mating efficiency when reconstituted with the wild-typeCPRαgene. Hyphal production on filament agar was not affected in theΔcprαstrain, indicating no significant role forCPRαin sensing environmental cues during haploid fruiting. The wild-typeMATα CPRαstrain produced abundant hyphae in response to syntheticMATa pheromone; however, the hyphal response to pheromone byΔcprαcells was significantly reduced. Exposure of wild-type cells to syntheticMATa pheromone for 2 h inducedMFαpheromone expression, whereas unexposed cells showed only basal levels of theMFαtranscript. TheΔcprαcells, however, exhibited only basal levels ofMFαmessage with or without pheromone exposure, suggesting thatCPRαandMFαare components of the same signaling pathway.
G-Protein Signaling Mediates Asexual Development at 25°C but Has No Effect on Yeast-Like Growth at 37°C in the Dimorphic Fungus
Penicillium marneffei
Authors: Zuber Sophie , Hynes Michael J. , Andrianopoulos Alex
Published: December 2002

The ascomycete
Penicillium marneffei
is an opportunistic human pathogen exhibiting a temperature-dependent dimorphic switch. At 25°C,
P. marneffei
grows as filamentous multinucleate hyphae and undergoes asexual development, producing uninucleate spores. At 37°C, it forms uninucleate yeast cells which divide by fission. We have cloned a gene encoding a Gα subunit of a heterotrimeric G protein from
P. marneffei
named
gasA
with high similarity to
fadA
in
Aspergillus nidulans
. Through the characterization of a Δ
gasA
strain and mutants carrying a dominant activating or a dominant interfering
gasA
allele, we show that GasA is a key regulator of asexual development but seems to play no role in the regulation of growth. A dominant activating
gasA
mutant whose mutation results in a G42-to-R change (
gasA
G42R
) does not express
brlA
, the conidiation-specific regulatory gene, and is locked in vegetative growth, while a dominant interfering
gasA
G203R
mutant shows inappropriate
brlA
expression and conidiation. Interestingly, the
gasA
mutants have no apparent defect in dimorphic switching or yeast-like growth at 37°C. Growth tests on dibutyryl cyclic AMP (dbcAMP) and theophylline suggest that a cAMP-protein kinase A cascade may be involved in the GasA signaling pathway.
Divergent Subunit Interactions among Fungal mRNA 5′-Capping Machineries
Authors: Takagi Toshimitsu , Cho Eun-Jung , Janoo Rozmin T. K. , Polodny Vladimir , Takase Yasutaka , Keogh Michael-C. , Woo Sue-Ann , Fresco-Cohen Lucille D. , Hoffman Charles S. , Buratowski Stephen
Published: December 2002

The
Saccharomyces cerevisiae
mRNA capping enzyme consists of two subunits: an RNA 5′-triphosphatase (RTPase) and GTP::mRNA guanylyltransferase (GTase). The GTase subunit (Ceg1) binds to the phosphorylated carboxyl-terminal domain of the largest subunit (CTD-P) of RNA polymerase II (pol II), coupling capping with transcription. Ceg1 bound to the CTD-P is inactive unless allosterically activated by interaction with the RTPase subunit (Cet1). For purposes of comparison, we characterize here the related GTases and RTPases from the yeasts
Schizosaccharomyces pombe
and
Candida albicans
. Surprisingly, the
S. pombe
capping enzyme subunits do not interact with each other. Both can independently interact with CTD-P of pol II, and the GTase is not repressed by CTD-P binding. The
S. pombe
RTPase gene (
pct1
+
) is essential for viability. Pct1 can replace the
S. cerevisiae
RTPase when GTase activity is supplied by the
S. pombe
or mouse enzymes but not by the
S. cerevisiae
GTase. The
C. albicans
capping enzyme subunits do interact with each other. However, this interaction is not essential in vivo. Our results reveal an unexpected diversity among the fungal capping machineries.
Saccharomyces cerevisiae
Cdc42p Localizes to Cellular Membranes and Clusters at Sites of Polarized Growth
Authors: Richman Tamara J. , Sawyer Mathew M. , Johnson Douglas I.
Published: December 2002

The Cdc42p GTPase controls polarized growth and cell cycle progression in eukaryotes from yeasts to mammals, and its precise subcellular localization is essential for its function. To examine the cell cycle-specific targeting of Cdc42p in living yeast cells, a green fluorescent protein (GFP)-Cdc42 fusion protein was used. In contrast to previous immunolocalization data, GFP-Cdc42p was found at the plasma membrane around the entire cell periphery and at internal vacuolar and nuclear membranes throughout the cell cycle, and it accumulated or clustered at polarized growth sites, including incipient bud sites and mother-bud neck regions. These studies also showed that C-terminal CAAX and polylysine domains were sufficient for membrane localization but not for clustering. Time-lapse fluorescence microscopy showed that GFP-Cdc42p clustered at the incipient bud site prior to bud emergence and at the mother-bud neck region postanaphase as a diffuse, single band and persisted as two distinct bands on mother and daughter cells following cytokinesis and cell separation. Initial clustering occurred immediately prior to actomyosin ring contraction and persisted postcontraction. These results suggest that Cdc42p targeting occurs through a novel mechanism of membrane localization followed by cell cycle-specific clustering at polarized growth sites.
GTPase-Activating Proteins for Cdc42
Authors: Smith Gregory R. , Givan Scott A. , Cullen Paul , Sprague George F.
Published: December 2002

The Rho-type GTPase, Cdc42, has been implicated in a variety of functions in the yeast life cycle, including septin organization for cytokinesis, pheromone response, and haploid invasive growth. A group of proteins called GTPase-activating proteins (GAPs) catalyze the hydrolysis of GTP to GDP, thereby inactivating Cdc42. At the time this study began, there was one known GAP, Bem3, and one putative GAP, Rga1, for Cdc42. We identified another putative GAP for Cdc42 and named it Rga2 (Rho GTPase-activating protein 2). We confirmed by genetic and biochemical criteria that Rga1, Rga2, and Bem3 act as GAPs for Cdc42. A detailed characterization of Rga1, Rga2, and Bem3 suggested that they regulate different subsets of Cdc42 function. In particular, deletion of the individual GAPs conferred different phenotypes. For example, deletion of
RGA1
, but not
RGA2
or
BEM3
, caused hyperinvasive growth. Furthermore, overproduction or loss of Rga1 and Rga2, but not Bem3, affected the two-hybrid interaction of Cdc42 with Ste20, a p21-activated kinase (PAK) kinase required for haploid invasive growth. These results suggest Rga1, and possibly Rga2, facilitate the interaction of Cdc42 with Ste20 to mediate signaling in the haploid invasive growth pathway. Deletion of
BEM3
resulted in cells with severe morphological defects not observed in
rga1
Δ or
rga2
Δ strains. These data suggest that Bem3 and, to a lesser extent, Rga1 and Rga2 facilitate the role of Cdc42 in septin organization. Thus, it appears that the GAPs play a role in modulating specific aspects of Cdc42 function. Alternatively, the different phenotypes could reflect quantitative rather than qualitative differences in GAP activity in the mutant strains.
Mga2p Processing by Hypoxia and Unsaturated Fatty Acids in
Saccharomyces cerevisiae
: Impact on LORE-Dependent Gene Expression
Authors: Jiang Yide , Vasconcelles Michael J. , Wretzel Sharon , Light Anne , Gilooly Laura , McDaid Kevin , Oh Chan-Seok , Martin Charles E. , Goldberg Mark A.
Published: December 2002

In
Saccharomyces cerevisiae
,
OLE1
encodes a Δ9 fatty acid desaturase, an enzyme that plays a critical role in maintaining the correct ratio of saturated to monounsaturated fatty acids in the cell membrane. Previous studies have demonstrated that (i)
OLE1
expression is repressed by unsaturated fatty acids (UFAs) and induced by low oxygen tension, (ii) a component of this regulation is mediated through the same low oxygen response element (LORE) in the
OLE1
promoter, and (iii) Mga2p is involved in LORE-dependent hypoxic induction of
OLE1
. We now report that LORE-
CYC1
basal promoter-
lacZ
fusion reporter assays demonstrate that UFAs repress the reporter expression under hypoxic conditions in a dose-dependent manner via LORE. Electrophoretic mobility shift assays show that UFAs repress the hypoxia-induced complex formation with LORE. Studies with a construct encoding a truncated form of Mga2p support the hypothesis that both hypoxia and UFA signals affect the processing of Mga2p and the UFA repression of
OLE1
hypoxic induction is mediated through Mga2p. Data from Western blot assays provide evidence that under normoxic conditions, Mga2p processing produces approximately equimolar levels of the membrane-bound and processed forms and is unaffected by UFAs. Hypoxic induction of
OLE1
, however, is associated with increased processing of the protein, resulting in an approximately fivefold increase in the soluble active form that is counteracted by exposure of the cells to unsaturated fatty acids. Data from this study suggest that the Mga2p-LORE interaction plays an important role in
OLE1
expression under both normoxic and hypoxic conditions.
Rub1p Processing by Yuh1p Is Required for Wild-Type Levels of Rub1p Conjugation to Cdc53p
Authors: Linghu Bolan , Callis Judy , Goebl Mark G.
Published: December 2002

In
Saccharomyces cerevisiae
, Rub1p, like ubiquitin, is conjugated to proteins. Before protein conjugation, the carboxyl-terminal asparagine residue of Rub1p is removed. Rub1p conjugation is dependent on the carboxyl-terminal processing enzyme Yuh1p, whereas Rub1p lacking the asparagine residue is conjugated without Yuh1p. Thus, Yuh1p is the major processing enzyme for Rub1p.
Two Copies of
mthmg1
, Encoding a Novel Mitochondrial HMG-Like Protein, Delay Accumulation of Mitochondrial DNA Deletions in
Podospora anserina
Authors: Dequard-Chablat Michelle , Alland Cynthia
Published: December 2002

In the filamentous fungus
Podospora anserina
, two degenerative processes which result in growth arrest are associated with mitochondrial genome (mitochondrial DNA [mtDNA]) instability. Senescence is correlated with mtDNA rearrangements and amplification of specific regions (senDNAs). Premature death syndrome is characterized by the accumulation of specific mtDNA deletions. This accumulation is due to indirect effects of the
AS1-4
mutation, which alters a cytosolic ribosomal protein gene. The
mthmg1
gene has been identified as a double-copy suppressor of premature death. It greatly delays premature death and the accumulation of deletions when it is present in two copies in an
AS1-4
context. The duplication of
mthmg1
has no significant effect on the wild-type life span or on senDNA patterns. In an
AS1
+
context, deletion of the
mthmg1
gene alters germination, growth, and fertility and reduces the life span. The
Δmthmg1
senescent strains display a particular senDNA pattern. This deletion is lethal in an
AS1-4
context. According to its physical properties (very basic protein with putative mitochondrial targeting sequence and HMG-type DNA-binding domains) and the cellular localization of an mtHMG1-green fluorescent protein fusion, mtHMG1 appears to be a mitochondrial protein possibly associated with mtDNA. It is noteworthy that it is the first example of a protein combining the two DNA-binding domains, AT-hook motif and HMG-1 boxes. It may be involved in the stability and/or transmission of the mitochondrial genome. To date, no structural homologues have been found in other organisms. However, mtHMG1 displays functional similarities with the
Saccharomyces cerevisiae
mitochondrial HMG-box protein Abf2.
Large-Scale Identification of Putative Exported Proteins in
Candida albicans
by Genetic Selection
Authors: Monteoliva L. , López Matas M. , Gil C. , Nombela C. , Pla J.
Published: December 2002

In all living organisms, secreted proteins play essential roles in different processes. Of special interest is the construction of the fungal cell wall, since this structure is absent from mammalian cells. The identification of the proteins involved in its biogenesis is therefore a primary goal in antifungal research. To perform a systematic identification of such proteins in
Candida albicans
, we carried out a genetic screening in which in-frame fusions with an intracellular allele of invertase gene
SUC2
of
Saccharomyces cerevisiae
can be used to select and identify putatively exported proteins in the heterologous host
S
.
cerevisiae
. Eighty-three clones were selected, including 11 previously identified genes from
C
.
albicans
as well as 41
C
.
albicans
genes that encode proteins homologous to already described proteins from related organisms. They include enzymes involved in cell wall synthesis and protein secretion. We also found membrane receptors and transporters presumably related to the interaction of
C
.
albicans
with the environment as well as extracellular enzymes and proteins involved in different morphological transitions. In addition, 11
C
.
albicans
open reading frames (ORFs) identified in this screening encode proteins homologous to unknown or putative proteins, while 5 ORFs encode novel secreted proteins without known homologues in other organisms. This screening procedure therefore not only identifies a set of targets of interest in antifungal research but also provides new clues for understanding the topological locations of many proteins involved in processes relevant to the pathogenicity of this microorganism.
Isolation and Characterization of
YlBEM1
, a Gene Required for Cell Polarization and Differentiation in the Dimorphic Yeast
Yarrowia lipolytica
Authors: Hurtado Cleofe A. R. , Rachubinski Richard A.
Published: December 2002

The ability to switch between a unicellular yeast form and different filamentous forms (fungal dimorphism) is an important attribute of most pathogenic fungi. Dimorphism involves a series of events that ultimately result in dramatic changes in the polarity of cell growth in response to environmental factors. We have isolated and characterized
YlBEM1
, a gene encoding a protein of 639 amino acids that is essential for the yeast-to-hypha transition in the yeast
Yarrowia lipolytica
and whose transcription is significantly increased during this event. Cells with deletions of
YlBEM1
are viable but show substantial alterations in morphology, disorganization of the actin cytoskeleton, delocalization of cortical actin and chitin deposition, multinucleation, and loss of mating ability, thus pointing to a major role for
YlBEM1
in the regulation of cell polarity and morphogenesis in this fungus. This role is further supported by the localization of YlBem1p, which, like cortical actin, appears to be particularly abundant at sites of growth of yeast, hyphal, and pseudohyphal cells. In addition, the potential involvement of YlBem1p in septum formation and/or cytokinesis is suggested by the concentration of a green fluorescent protein-tagged version of this protein at the mother-bud neck during the last stages of cell division. Interestingly, overexpression of
MHY1
,
YlRAC1
, or
YlSEC31
, three genes involved in filamentous growth of
Y. lipolytica
, induced hyphal growth of
bem1
null mutant cells.
Characterization of Inhibitor-Resistant Histone Deacetylase Activity in Plant-Pathogenic Fungi
Authors: Baidyaroy Dipnath , Brosch Gerald , Graessle Stefan , Trojer Patrick , Walton Jonathan D.
Published: December 2002

HC-toxin, a cyclic peptide made by the filamentous fungus
Cochliobolus carbonum
, is an inhibitor of histone deacetylase (HDAC) from many organisms. It was shown earlier that the HDAC activity in crude extracts of
C. carbonum
is relatively insensitive to HC-toxin as well as to the chemically unrelated HDAC inhibitors trichostatin and D85, whereas the HDAC activity of
Aspergillus nidulans
is sensitive (G. Brosch et al., Biochemistry
40:
12855-12863, 2001). Here we report that HC-toxin-resistant HDAC activity was present in other, but not all, plant-pathogenic
Cochliobolus
species but not in any of the saprophytic species tested. The HDAC activities of the fungi
Alternaria brassicicola
and
Diheterospora chlamydosporia
, which also make HDAC inhibitors, were resistant. The HDAC activities of all
C. carbonum
isolates tested, except one non-toxin-producing isolate, were resistant. In a cross between a sensitive isolate and a resistant isolate, resistance genetically cosegregated with HC-toxin production. When fractionated by anion-exchange chromatography, extracts of resistant and sensitive isolates and species had two peaks of HDAC activity, one that was fully HC-toxin resistant and a second that was larger and sensitive. The first peak was consistently smaller in extracts of sensitive fungi than in resistant fungi, but the difference appeared to be insufficiently large to explain the differential sensitivities of the crude extracts. Differences in mRNA expression levels of the four known HDAC genes of
C. carbonum
did not account for the observed differences in HDAC activity profiles. When mixed together, resistant extracts protected extracts of sensitive
C. carbonum
but did not protect other sensitive
Cochlibolus
species or
Neurospora crassa
. Production of this extrinsic protection factor was dependent on
TOXE
, the transcription factor that regulates the HC-toxin biosynthetic genes. The results suggest that
C. carbonum
has multiple mechanisms of self-protection against HC-toxin.
Functional Diversity of Silencers in Budding Yeasts
Authors: Sjöstrand Jimmy O. O. , Kegel Andreas , Åström Stefan U.
Published: December 2002

We studied the silencing of the cryptic mating-type loci
HML
α and
HMR
a in the budding yeast
Kluyveromyces lactis
. A 102-bp minimal silencer fragment was defined that was both necessary and sufficient for silencing of
HML
α. Mutagenesis of the silencer revealed three distinct regions (A, B, and C) that were important for silencing. Recombinant
K. lactis
ribosomal DNA enhancer binding protein 1 (Reb1p) could bind the silencer in vitro, and point mutations in the B box abolished both Reb1p binding and silencer function. Furthermore, strains carrying temperature-sensitive alleles of the
REB1
gene derepressed the transcription of the
HML
α
1
gene at the nonpermissive temperature. A functional silencer element from the
K. lactis
cryptic
HMR
a locus was also identified, which contained both Reb1p binding sites and A boxes, strongly suggesting a general role for these sequences in
K. lactis
silencing. Our data indicate that different proteins bind to
Kluyveromyces
silencers than to
Saccharomyces
silencers. We suggest that the evolution of silencers is rapid in budding yeasts and discuss the similarities and differences between silencers in
Saccharomyces
and
Kluyveromyces
.
Schizosaccharomyces pombe
Git7p, a Member of the
Saccharomyces cerevisiae
Sgt1p Family, Is Required for Glucose and Cyclic AMP Signaling, Cell Wall Integrity, and Septation
Authors: Schadick Kevin , Fourcade H. Matthew , Boumenot Peter , Seitz Jeffrey J. , Morrell Jennifer L. , Chang Louise , Gould Kathleen L. , Partridge Janet F. , Allshire Robin C. , Kitagawa Katsumi , Hieter Phil , Hoffman Charles S.
Published: December 2002

The
Schizosaccharomyces pombe fbp1
gene, encoding fructose-1,6-bisphosphatase, is transcriptionally repressed by glucose. Mutations that confer constitutive
fbp1
transcription identify
git
(glucose-insensitive transcription) genes that encode components of a cyclic AMP (cAMP) signaling pathway required for adenylate cyclase activation. Four of these genes encode the three subunits of a heterotrimeric G protein (
gpa2
,
git5
, and
git11
) and a G protein-coupled receptor (
git3
). Three additional genes,
git1
,
git7
, and
git10
, act in parallel to or downstream from the G protein genes. Here, we describe the cloning and characterization of the
git7
gene. The Git7p protein is a member of the
Saccharomyces cerevisiae
Sgt1p protein family. In budding yeast, Sgt1p associates with Skp1p and plays an essential role in kinetochore assembly, while in
Arabidopsis
, a pair of SGT1 proteins have been found to be involved in plant disease resistance through an interaction with RAR1. Like
S
.
cerevisiae
Sgt1p, Git7p is essential, but this requirement appears to be due to roles in septation and cell wall integrity, which are unrelated to cAMP signaling, as
S
.
pombe
cells lacking either adenylate cyclase or protein kinase A are viable. In addition,
git7
mutants are sensitive to the microtubule-destabilizing drug benomyl, although they do not display a chromosome stability defect. Two alleles of
git7
that are functional for cell growth and septation but defective for glucose-triggered cAMP signaling encode proteins that are altered in the highly conserved carboxy terminus. The
S
.
cerevisiae
and human
SGT1
genes both suppress
git7-93
but not
git7-235
for glucose repression of
fbp1
transcription and benomyl sensitivity. This allele-specific suppression indicates that the Git7p/Sgt1p proteins may act as multimers, such that Git7-93p but not Git7-235p can deliver the orthologous proteins to species-specific targets. Our studies suggest that members of the Git7p/Sgt1p protein family may play a conserved role in the regulation of adenylate cyclase activation in
S
.
pombe
,
S
.
cerevisiae
, and humans.
Sgt1p Contributes to Cyclic AMP Pathway Activity and Physically Interacts with the Adenylyl Cyclase Cyr1p/Cdc35p in Budding Yeast
Authors: Dubacq Caroline , Guerois Raphaël , Courbeyrette Régis , Kitagawa Katsumi , Mann Carl
Published: December 2002

Sgt1p is a highly conserved eucaryotic protein that is required for both SCF (Skp1p/Cdc53p-Cullin-F-box)-mediated ubiquitination and kinetochore function in yeast. We show here that Sgt1p is also involved in the cyclic AMP (cAMP) pathway in
Saccharomyces cerevisiae
.
SGT1
is an allele-specific suppressor of
cdc35-1
, a thermosensitive mutation in the leucine-rich repeat domain of the adenylyl cyclase Cyr1p/Cdc35p. We demonstrate that Sgt1p and Cyr1p/Cdc35p physically interact and that the activity of the cAMP pathway is affected in an
sgt1
conditional mutant. Sequence analysis suggests that Sgt1p has features of a cochaperone. Thus, Sgt1p is a novel activator of adenylyl cyclase in
S
.
cerevisiae
and may function in the assembly or the conformational activation of specific multiprotein complexes.
New Class of Cargo Protein inTetrahymena thermophilaDense Core Secretory Granules
Authors: Haddad Alex , Bowman Grant R. , Turkewitz Aaron P.
Published: December 2002

Regulated exocytosis of dense core secretory granules releases biologically active proteins in a stimulus-dependent fashion. The packaging of the cargo within newly forming granules involves a transition: soluble polypeptides condense to form water-insoluble aggregates that constitute the granule cores. Following exocytosis, the cores generally disassemble to diffuse in the cell environment. The ciliatesTetrahymena thermophilaandParamecium tetraureliahave been advanced as genetically manipulatable systems for studying exocytosis via dense core granules. However, all of the known granule proteins in these organisms condense to form the architectural units of lattices that are insoluble both before and after exocytosis. Using an approach designed to detect new granule proteins, we have now identified Igr1p (induced during granule regeneration). By structural criteria, it is unrelated to the previously characterized lattice-forming proteins. It is distinct in that it is capable of dissociating from the insoluble lattice following secretion and therefore represents the first diffusible protein identified in ciliate granules.
Trichoderma atroviride
G-Protein α-Subunit Gene
tga1
Is Involved in Mycoparasitic Coiling and Conidiation
Authors: Rocha-Ramírez Víctor , Omero Carmi , Chet Ilan , Horwitz Benjamin A. , Herrera-Estrella Alfredo
Published: December 2002

The soil fungus
Trichoderma atroviride
, a mycoparasite, responds to a number of external stimuli. In the presence of a fungal host,
T
.
atroviride
produces hydrolytic enzymes and coils around the host hyphae. In response to light or nutrient depletion, asexual sporulation is induced. In a biomimetic assay, different lectins induce coiling around nylon fibers; coiling in the absence of lectins can be induced by applying cyclic AMP (cAMP) or the heterotrimeric G-protein activator mastoparan. We isolated a
T
.
atroviride
G-protein α-subunit (Gα) gene (
tga1
) belonging to the fungal subfamily with the highest similarity to the Gα
i
class. Generated transgenic lines that overexpress Gα show very delayed sporulation and coil at a higher frequency. Furthermore, transgenic lines that express an activated mutant protein with no GTPase activity do not sporulate and coil at a higher frequency. Lines that express an antisense version of the gene are hypersporulating and coil at a much lower frequency in the biomimetic assay. The loss of Tga1 in these mutants correlates with the loss of GTPase activity stimulated by the peptide toxin Mas-7. The application of Mas-7 to growing mycelial colonies raises intracellular cAMP levels, suggesting that Tga1 can activate adenylyl cyclase. In contrast, cAMP levels and cAMP-dependent protein kinase activity drop when diffusible host signals are encountered and the mycoparasitism-related genes
ech42
and
prb1
are highly expressed. Mycoparasitic signaling is unlikely to be a linear pathway from host signals to increased cAMP levels. Our results demonstrate that the product of the
tga1
gene is involved in both coiling and conidiation.
Interaction with Btn2p Is Required for Localization of Rsg1p: Btn2p-Mediated Changes in Arginine Uptake in
Saccharomyces cerevisiae
Authors: Chattopadhyay Subrata , Pearce David A.
Published: December 2002

Btn2p, a novel coiled-coil protein, is up-regulated in
btn1
Δ yeast strains, and this up-regulation is thought to contribute to maintaining a stable vacuolar pH in
btn1
Δ strains (D. A. Pearce, T. Ferea, S. A. Nosel, B. Das, and F. Sherman, Nat. Genet.
22:
55-58, 1999). We now report that Btn2p interacts biochemically and functionally with Rsg1p, a down-regulator of the Can1p arginine and lysine permease. Rsg1p localizes to a distinct structure toward the cell periphery, and strains lacking Btn2p (
btn2
Δ strains) fail to correctly localize Rsg1p.
btn2
Δ strains, like
rsg1
Δ strains, are sensitive for growth in the presence of the arginine analog canavanine. Furthermore,
btn2
Δ strains, like
rsg1
Δ strains, demonstrate an elevated rate of uptake of [
14
C]arginine, which leads to increased intracellular levels of arginine. Overexpression of
BTN2
results in a decreased rate of arginine uptake. Collectively, these results indicate that altered levels of Btn2p can modulate arginine uptake through localization of the Can1p-arginine permease regulatory protein, Rsg1p. Our original identification of Btn2p was that it is up-regulated in the
btn1
Δ strain which serves as a model for the lysosomal storage disorder Batten disease. Btn1p is a vacuolar/lysosomal membrane protein, and
btn1
Δ suppresses both the canavanine sensitivity and the elevated rate of uptake of arginine displayed by
btn2
Δ
rsg1
Δ strains. We conclude that Btn2p interacts with Rsg1p and modulates arginine uptake. Up-regulation of
BTN2
expression in
btn1
Δ strains may facilitate either a direct or indirect effect on intracellular arginine levels.
The Fission Yeast Ubiquitin-Conjugating Enzymes UbcP3, Ubc15, and Rhp6 Affect Transcriptional Silencing of the Mating-Type Region
Authors: Sig Nielsen Inga , Nielsen Olaf , Murray Johanne M. , Thon Geneviève
Published: December 2002

Genes transcribed by RNA polymerase II are silenced when introduced near the
mat2
or
mat3
mating-type loci of the fission yeast
Schizosaccharomyces pombe
. Silencing is mediated by a number of gene products and
cis
-acting elements. We report here the finding of novel
trans
-acting factors identified in a screen for high-copy-number disruptors of silencing. Expression of cDNAs encoding the putative E2 ubiquitin-conjugating enzymes UbcP3, Ubc15 (ubiquitin-conjugating enzyme), or Rhp6 (Rad homolog
pombe
) from the strong
nmt1
promoter derepressed the silent mating-type loci
mat2
and
mat3
and reporter genes inserted nearby. Deletion of
rhp6
slightly derepressed an
ade6
reporter gene placed in the mating-type region, whereas disruption of
ubcP3
or
ubc15
had no obvious effect on silencing. Rhp18 is the
S. pombe
homolog of
Saccharomyces cerevisiae
Rad18p, a DNA-binding protein that physically interacts with Rad6p. Rhp18 was not required for the derepression observed when UbcP3, Ubc15, or Rhp6 was overproduced. Overexpressing Rhp6 active-site mutants showed that the ubiquitin-conjugating activity of Rhp6 is essential for disruption of silencing. However, high dosage of UbcP3, Ubc15, or Rhp6 was not suppressed by a mutation in the 26S proteasome, suggesting that loss of silencing is not due to an increased degradation of silencing factors but rather to the posttranslational modification of proteins by ubiquitination. We discuss the implications of these results for the possible modes of action of UbcP3, Ubc15, and Rhp6.
Colletotrichum trifolii
TB3 Kinase, a COT1 Homolog, Is Light Inducible and Becomes Localized in the Nucleus during Hyphal Elongation
Authors: Chen Changbin , Dickman Martin B.
Published: December 2002

Colletotrichum trifolii
is a fungal pathogen responsible for anthracnose disease of alfalfa. Previously, a serine/threonine protein kinase gene from this fungus (TB3), which is a functional homolog of the
Neurospora crassa
COT1 kinase, has been isolated in our laboratory and appears to be associated with hyphal elongation and branching. In this report we show that light treatment rapidly induces TB3 expression and hyphal branching frequency. Western analysis showed TB3 localization in both the cytoplasm and nucleus, but not in membranes. Moreover, indirect immunofluorescence indicated that TB3 levels were most abundant in the nucleus. To further evaluate the subcellular distribution of TB3, a TB3::GFP fusion construct was inserted into
C. trifolii
. Results indicated that the cellular location of TB3 changed during fungal growth and development. Consistent with previous observations, TB3 was localized in both the cytoplasm and the nucleus but was preferentially localized in the nucleus during extended hyphal growth. The amino terminus of TB3 contains two relatively long polyglutamine repeats. Yeast-based assays showed that these polyglutamine tracts can activate transcription. These results suggest that TB3 may be positioned in a signaling cascade regulating proper hyphal growth and development by functioning as a transcription factor.
Shared and Independent Roles for a Gα
i
Protein and Adenylyl Cyclase in Regulating Development and Stress Responses in
Neurospora crassa
Authors: Ivey F. Douglas , Kays Ann M. , Borkovich Katherine A.
Published: December 2002

Growth and development are regulated using cyclic AMP (cAMP)-dependent and -independent pathways in
Neurospora crassa
. The
cr-1
adenylyl cyclase mutant lacks detectable cAMP and exhibits numerous defects, including colonial growth habit, short aerial hyphae, premature conidiation on plates, inappropriate conidiation in submerged culture, and increased thermotolerance. Evidence suggests that the heterotrimeric Gα protein GNA-1 is a direct positive regulator of adenylyl cyclase. Δ
gna-1
strains are female-sterile, and Δ
gna-1
strains have reduced apical extension rates on normal and hyperosmotic medium, greater resistance to oxidative and heat stress, and stunted aerial hyphae compared to the wild-type strain. In this study, a Δ
gna-1 cr-1
double mutant was analyzed to differentiate cAMP-dependent and -independent signaling pathways regulated by GNA-1. Δ
gna-1 cr-1
mutants have severely restricted colonial growth and do not produce aerial hyphae on plates or in standing liquid cultures. Addition of cAMP to plates or standing liquid cultures rescues
cr-1
, but not Δ
gna-1 cr-1
, defects, which is consistent with previous results demonstrating that Δ
gna-1
mutants do not respond to exogenous cAMP. The females of all strains carrying the Δ
gna-1
mutation are sterile; however, unlike
cr-1
and Δ
gna-1
strains, the Δ
gna-1 cr-1
mutant does not produce protoperithecia. The Δ
gna-1
and
cr-1
mutations were synergistic with respect to inappropriate conidiation during growth in submerged culture. Thermotolerance followed the order wild type < Δ
gna-1
<
cr-1
= Δ
gna-1 cr-1
, consistent with a cAMP-dependent process. Taken together, the results suggest that in general, GNA-1 and CR-1 regulate
N. crassa
growth and development using parallel pathways, while thermotolerance is largely dependent on cAMP.
Evolutionary Analyses of ABC Transporters of
Dictyostelium discoideum
Authors: Anjard Christophe ,   the Dictyostelium Sequencing Consortium, Loomis William F.
Published: December 2002

The ABC superfamily of genes is one of the largest in the genomes of both bacteria and eukaryotes. The proteins encoded by these genes all carry a characteristic 200- to 250-amino-acid ATP-binding cassette that gives them their family name. In bacteria they are mostly involved in nutrient import, while in eukaryotes many are involved in export. Seven different families have been defined in eukaryotes based on sequence homology, domain topology, and function. While only 6 ABC genes in
Dictyostelium discoideum
have been studied in detail previously, sequences from the well-advanced
Dictyostelium
genome project have allowed us to recognize 68 members of this superfamily. They have been classified and compared to animal, plant, and fungal orthologs in order to gain some insight into the evolution of this superfamily. It appears that many of the genes inferred to have been present in the ancestor of the crown organisms duplicated extensively in some but not all phyla, while others were lost in one lineage or the other.
Synthesis of Chloroplast Galactolipids in Apicomplexan Parasites
Authors: Maréchal Eric , Azzouz Nahid , Santos de Macedo Cristiana , Block Maryse A. , Feagin Jean E. , Schwarz Ralph T. , Joyard Jacques
Published: December 2002

Monogalactosyldiacylglycerol and digalactosyldiacylglycerol are major chloroplast lipids of algae and land plants and are synthesized within the plastid envelope. Here we report that in
Toxoplasma gondii
and
Plasmodium falciparum
lysates, radiolabeled UDP-galactose is incorporated into monogalactosylcerebrosides, monogalactosyldiacylglycerol, and digalactosyldiacylglycerol due to distinct enzymological activities. Furthermore, DGDG is immunologically detected in apicomplexans.
Amino Acid-Dependent Gcn4p Stability Regulation Occurs Exclusively in the Yeast Nucleus
Authors: Pries Ralph , Bömeke Katrin , Irniger Stefan , Grundmann Olav , Braus Gerhard H.
Published: December 2002

The c-Jun-like transcriptional activator Gcn4p controls biosynthesis of translational precursors in the yeast
Saccharomyces cerevisiae
. Protein stability is dependent on amino acid limitation and
cis
signals within Gcn4p which are recognized by cyclin-dependent protein kinases, including Pho85p. The Gcn4p population within unstarved yeast consists of a small relatively stable cytoplasmic fraction and a larger less stable nuclear fraction. Gcn4p contains two nuclear localization signals (NLS) which function independently of the presence or absence of amino acids. Expression of NLS-truncated Gcn4p results in an increased cytoplasmic fraction and an overall stabilization of the protein. The same effect is achieved for the entire Gcn4p in a
yrb1
yeast mutant strain impaired in the nuclear import machinery. In the presence of amino acids, controlled destabilization of Gcn4p is triggered by the phosphorylation activity of Pho85p. A
pho85Δ
mutation stabilizes Gcn4p without affecting nuclear import. Pho85p is localized within the nucleus in the presence or absence of amino acids. Therefore, there is a strict spatial separation of protein synthesis and degradation of Gcn4p in yeast. Control of protein stabilization which antagonizes Gcn4p function is restricted to the nucleus.
Dual Role of the
Saccharomyces cerevisiae
TEA/ATTS Family Transcription Factor Tec1p in Regulation of Gene Expression and Cellular Development
Authors: Köhler Tim , Wesche Stefanie , Taheri Naimeh , Braus Gerhard H. , Mösch Hans-Ulrich
Published: December 2002

In
Saccharomyces cerevisiae
, the transcription factors Tec1p and Ste12p are required for haploid invasive and diploid pseudohyphal growth. Tec1p and Ste12p have been postulated to regulate these developmental processes primarily by cooperative binding to filamentous and invasion-responsive elements (FREs), which are combined enhancer elements that consist of a Tec1p-binding site (TCS) and an Ste12p-binding site (PRE). They are present in the promoter regions of target genes, e.g.,
FLO11
. Here, we show that Tec1p efficiently activates target gene expression and cellular development in the absence of Ste12p. We further demonstrate that TCS elements alone are sufficient to mediate Tec1p-driven gene expression by a mechanism termed TCS control that is operative even when Ste12p is absent. Mutational analysis of
TEC1
revealed that TCS control,
FLO11
expression, and haploid invasive growth require the C terminus of Tec1p. In contrast, the Ste12p-dependent FRE control mechanism is sufficiently executed by the N-terminal portion of Tec1p, which contains the TEA/ATTS DNA-binding domain. Our study suggests that regulation of haploid invasive and diploid pseudohyphal growth by Ste12p and Tec1p is not only executed by combinatorial control but involves additional control mechanisms in which Ste12p activates
TEC1
expression via clustered PREs and where Tec1p regulates expression of target genes, e.g.,
FLO11
, by TCS control.
Novel Protein Kinase Induced during Sporangial Cleavage in the Oomycete
Phytophthora infestans
Authors: Judelson Howard S. , Roberts Samuel
Published: December 2002

A study of the effect of inhibitors on zoospore development in
Phytophthora infestans
demonstrated the involvement of protein kinases and calcium and led to the discovery of a gene induced during zoosporogenesis that encoded a protein resembling Ca
+2
- and calmodulin-regulated serine/threonine protein kinases. The calcium channel blocker verapamil and the calmodulin antagonist trifluoroperazine inhibited zoosporogenesis and encystment. The protein kinase inhibitors K-252a and KN-93 inhibited zoospore release, encystment, and cyst germination, and K-252a reduced zoospore viability. In contrast, the inhibitors had minor or no effects on sporangia directly germinating in media. Spurred by these findings, a survey of putative protein kinase genes was performed to identify any that were up-regulated during zoosporogenesis. A kinase-encoding gene was identified for which mRNA accumulation was first detected soon after chilling sporangia in water, conditions that induce sporangial cytoplasm to cleave and release zoospores. The transcript persisted in motile zoospores and in germinated cysts but was not detected in other tissues, including hyphae, hyphae placed in water, or directly germinating sporangia. The structure of the predicted protein was novel, as its C-terminal region, which binds calmodulin in related proteins, was unusually short. Concentrations of actinomycin D previously used in experiments that suggested that de novo transcription was not needed for zoosporogenesis or encystment only partially inhibited transcription of the kinase gene, probably due to poor uptake into sporangia.
Intracellular Maltose Is Sufficient To Induce
MAL
Gene Expression in
Saccharomyces cerevisiae
Authors: Wang Xin , Bali Mehtap , Medintz Igor , Michels Corinne A.
Published: December 2002

The presence of maltose induces
MAL
gene expression in
Saccharomyces
cells, but little is known about how maltose is sensed. Strains with all maltose permease genes deleted are unable to induce
MAL
gene expression. In this study, we examined the role of maltose permease in maltose sensing by substituting a heterologous transporter for the native maltose permease.
PmSUC2
encodes a sucrose transporter from the dicot plant
Plantago major
that exhibits no significant sequence homology to maltose permease. When expressed in
Saccharomyces cerevisiae
, PmSUC2 is capable of transporting maltose, albeit at a reduced rate. We showed that introduction of
PmSUC2
restores maltose-inducible
MAL
gene expression to a maltose permease-null mutant and that this induction requires the
MAL
activator. These data indicate that intracellular maltose is sufficient to induce
MAL
gene expression independently of the mechanism of maltose transport. By using strains expressing defective
mal61
mutant alleles, we demonstrated a correlation between the rate of maltose transport and the level of the induction, which is particularly evident in medium containing very limiting concentrations of maltose. Moreover, our results indicate that a rather low concentration of intracellular maltose is needed to trigger
MAL
gene expression. We also showed that constitutive overexpression of either
MAL61
maltose permease or
PmSUC2
suppresses the noninducible phenotype of a defective
mal13 MAL
-activator allele, suggesting that this suppression is solely a function of maltose transport activity and is not specific to the sequence of the permease. Our studies indicate that maltose permease does not function as the maltose sensor in
S. cerevisiae
.
Mating-Type Locus of
Cryptococcus neoformans
: a Step in the Evolution of Sex Chromosomes
Authors: Lengeler Klaus B. , Fox Deborah S. , Fraser James A. , Allen Andria , Forrester Keri , Dietrich Fred S. , Heitman Joseph
Published: December 2002

The sexual development and virulence of the fungal pathogen
Cryptococcus neoformans
is controlled by a bipolar mating system determined by a single locus that exists in two alleles, α and
a
. The α and
a
mating-type alleles from two divergent varieties were cloned and sequenced. The
C. neoformans
mating-type locus is unique, spans >100 kb, and contains more than 20 genes.
MAT
-encoded products include homologs of regulators of sexual development in other fungi, pheromone and pheromone receptors, divergent components of a MAP kinase cascade, and other proteins with no obvious function in mating. The α and
a
alleles of the mating-type locus have extensively rearranged during evolution and strain divergence but are stable during genetic crosses and in the population. The
C. neoformans
mating-type locus is strikingly different from the other known fungal mating-type loci, sharing features with the self-incompatibility systems and sex chromosomes of algae, plants, and animals. Our study establishes a new paradigm for mating-type loci in fungi with implications for the evolution of cell identity and self/nonself recognition.
Isocitrate Lyase Is Essential for Pathogenicity of the Fungus
Leptosphaeria maculans
to Canola
(
Brassica napus
)
Authors: Idnurm Alexander , Howlett Barbara J.
Published: December 2002

A pathogenicity gene has been identified in
Leptosphaeria maculans
, the ascomycetous fungus that causes blackleg disease of canola (
Brassica napus)
. This gene encodes isocitrate lyase, a component of the glyoxylate cycle, and is essential for the successful colonization of
B. napus
. It was identified by a reverse genetics approach whereby a plasmid conferring hygromycin resistance was inserted randomly into the
L. maculans
genome. Twelve of 516 transformants tested had reduced pathogenicity on cotyledons of
B. juncea
and
B. napus
, and 1 of these 12 had a deletion of the isocitrate lyase gene, as well as an insertion of the hygromycin resistance gene. This mutant was unable to grow on fatty acids, including monolaurate, and the isocitrate lyase transcript was not detected. When the wild-type gene was reintroduced into the mutant, growth on monolaurate was restored and pathogenicity was partially restored.
L. maculans
isocitrate lyase is produced during infection of
B. napus
cotyledons, while the plant homologue is not. When 2.5% glucose was added to the inoculum of the isocitrate lyase mutant, lesions of sizes similar to those caused by wild-type isolate M1 developed on
B. napus
cotyledons. These findings suggest that the glyoxylate pathway is essential for disease development by this plant-pathogenic fungus, as has been shown recently for a fungal and bacterial pathogen of animals and a bacterial pathogen of plants. Involvement of the glyoxylate pathway in pathogenesis in animals and plants presents potential drug targets for control of diseases.
Aspergillus nidulansCatalase-Peroxidase Gene (cpeA) Is Transcriptionally Induced during Sexual Development through the Transcription Factor StuA
Authors: Scherer Mario , Wei Huijun , Liese Ralf , Fischer Reinhard
Published: December 2002

Catalases, peroxidases, and catalase-peroxidases are important enzymes to cope with reactive oxygen species in pro- and eukaryotic cells. In the filamentous fungusAspergillus nidulansthree monofunctional catalases have been described, and a fourth catalase activity was observed in native polyacrylamide gels. The latter activity is probably due to the bifunctional enzyme catalase-peroxidase, which we characterized here. The gene, namedcpeA, encodes an 81-kDa polypeptide with a conserved motif for heme coordination. The enzyme comprises of two similar domains, suggesting gene duplication and fusion during evolution. The first 439 amino acids share 22% identical residues with the C terminus. Homologous proteins are found in several prokaryotes, such asEscherichia coliandMycobacterium tuberculosis(both with 61% identity). In fungi the enzyme has been noted inPenicillium simplicissimum,Septoria tritici, andNeurospora crassa(69% identical amino acids) but is absent fromSaccharomyces cerevisiae.Expression analysis inA. nidulansrevealed that the gene is transcriptionally induced upon carbon starvation and during sexual development, but starvation is not sufficient to reach high levels of the transcript during development. Besides transcriptional activation, we present evidence for posttranscriptional regulation. A green fluorescent protein fusion protein localized to the cytoplasm of Hülle cells. The Hülle cell-specific expression was dependent on the developmental regulator StuA, suggesting an activating function of this helix-loop-helix transcription factor.