Isolation and Characterization of theCandida albicans PFY1 Gene for Profilin

Ostrander, Darin; Gorman, Jessica A.

doi:10.1002/(sici)1097-0061(199707)13:9<871::aid-yea127>3.0.co;2-2

Cited by 4 publications

(4 citation statements)

References 49 publications

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“…Because of problems associated with the stability of PGS1 plasmids in E. coli, ligations were transformed into and plasmids stored in yeast. Yeast spheroplast transformations of pgs1⌬ strains were accomplished as described (41). Transformants were selected on yeast nitrogen base uracil dropout medium containing ethanol and glycerol (36).…”

Section: Methodsmentioning

confidence: 99%

Lack of Mitochondrial Anionic Phospholipids Causes an Inhibition of Translation of Protein Components of the Electron Transport Chain

Ostrander¹,

Zhang

Mileykovskaya

et al. 2001

Journal of Biological Chemistry

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173

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Reduction of mitochondrial cardiolipin (CL) levels has been postulated to compromise directly the function of several essential enzymes and processes of the mitochondria. There is limited genetic evidence for the critical roles with which CL and its precursor phosphatidylglycerol (PG) have been associated. A null allele of the PGS1 gene from Saccharomyces cerevisiae, which encodes the enzyme responsible for the synthesis of the CL precursor PG phosphate, was created in a yeast strain in which PGS1 expression is exogenously regulated by doxycycline. The addition of increasing concentrations of doxycycline to the growth medium causes a proportional decrease to undetectable levels of PGS1 transcript, PG phosphate synthase activity, and PG plus CL. The doubling time of this strain with increasing doxycycline increases to senescence in non-fermentable carbon sources or at high temperatures, conditions that do not support growth of the pgs1⌬ strain. Doxycycline addition also causes mitochondrial abnormalities as observed by fluorescence microscopy. Products of four mitochondrial encoded genes (COX1, COX2, COX3, and COB) and one nuclear encoded gene (COX4) associated with the mitochondrial inner membrane are not present when PGS1 expression is fully repressed. No translation of these proteins can be detected in cells lacking the PGS1 gene product, although transcription and splicing appear unaffected. Protein import of other nuclear encoded proteins remains unaffected. The remaining proteins encoded by mitochondrial DNA are expressed and translated normally. Thus, the molecular basis for the lack of mitochondrial function in pgs1⌬ cells is the failure to translate gene products essential to the electron transport chain.

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Section: Methodsmentioning

confidence: 99%

Lack of Mitochondrial Anionic Phospholipids Causes an Inhibition of Translation of Protein Components of the Electron Transport Chain

Ostrander¹,

Zhang

Mileykovskaya

et al. 2001

Journal of Biological Chemistry

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173

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“…Likewise, the actin monomer recruiter profilin was not required. C. albicans profilin has been shown to be functionally similar to S. cerevisiae profilin (59), and the two proteins share extensive sequence homology (87% similarity, 72% identity). The pfy1⌬/pfy1⌬ strain showed alterations to hyphal structure similar to those observed in the bni1⌬/ bni1⌬ strain (not shown); however, HWP1 gene expression was not reduced in pfy1⌬/pfy1⌬ cells (Fig.…”

Section: Fig 2 (A)mentioning

confidence: 99%

Role of Actin Cytoskeletal Dynamics in Activation of the Cyclic AMP Pathway and HWP1 Gene Expression in Candida albicans

Wolyniak

Sundstrom

2007

Eukaryot Cell

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Changes in gene expression during reversible bud-hypha transitions of the opportunistic fungal pathogen Candida albicans permit adaptation to environmental conditions that are critical for proliferation in host tissues. Our previous work has shown that the hypha-specific adhesin gene HWP1 is up-regulated by the cyclic AMP (cAMP) signaling pathway. However, little is known about the potential influences of determinants of cell morphology on HWP1 gene expression. We found that blocking hypha formation with cytochalasin A, which destabilizes actin filaments, and with latrunculin A, which sequesters actin monomers, led to a loss of HWP1 gene expression. In contrast, high levels of HWP1 gene expression were observed when the F-actin stabilizer jasplakinolide was used to block hypha formation, suggesting that HWP1 expression could be regulated by actin structures. Mutants defective in formin-mediated nucleation of F-actin were reduced in HWP1 gene expression, providing genetic support for the importance of actin structures. Kinetic experiments with wildtype and actin-deficient cells revealed two distinct phases of HWP1 gene expression, with a slow, actinindependent phase preceding a fast, actin-dependent phase. Low levels of HWP1 gene expression that appeared to be independent of stabilized actin and cAMP signaling were detected using indirect immunofluorescence. A connection between actin structures and the cAMP signaling pathway was shown using hyper-and hypomorphic cAMP mutants, providing a possible mechanism for up-regulation of HWP1 gene expression by stabilized actin. The results reveal a new role for F-actin as a regulatory agent of hypha-specific gene expression at the bud-hypha transition.Candida albicans is an opportunistic fungal pathogen with a tropism for the gastrointestinal mucosa. The progression to candidiasis from the usual state of commensalism is prompted by changes in the immune status of the host that relay signals for proliferation and culminate in tissue invasion. Growth in host tissues is characterized by the presence of yeast, pseudohyphal, and hyphal morphologies in response to a number of environmental parameters within the host that may include temperature, pH, and nutrition (31,33,66) as well as other unknown factors. The different morphologies of C. albicans are accompanied by differences in gene expression patterns that are critical to the development of candidiasis. The mechanisms that mediate changes in gene expression in concert with changes in morphology are directly relevant to the regulation of virulence of C. albicans.The hypha-specific gene HWP1 (hyphal wall protein 1) is an attractive candidate for studying morphology-specific gene regulation since it is abundantly expressed in hyphal growth mode and is barely detectable in the yeast morphology (73, 74). Pseudohyphae vary in the level of HWP1 gene expression (71). HWP1 encodes a glycosylphosphatidylinositol-linked cell wall protein that serves as a transglutaminase substrate and enables C. albicans to form covalent attachments ...

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“…Strains used to test for pfy1 Δ lethality are listed in Table 1[19,29–31]. The construction of a PFY1 / pfy1 Δ strain and the yeast PFY1 expression plasmid has been previously described [31].…”

Section: Methodsmentioning

confidence: 99%

“…In the yeast Saccharomyces cerevisiae , the gene for profilin, PFY1 , has been cloned and characterized [18]. Deletion of the gene is a lethal event in many strains [18,19], while in others, the disruption produces strains with highly abnormal morphologies and severely retarded growth rates [20]. The effects of yeast profilin on actin binding and polymerization have been studied and found to be very similar to those of multicellular organisms [21].…”

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confidence: 99%

Polyproline binding is an essential function of human profilin in yeast

Ostrander

Ernst

Lavoie

et al. 1999

European Journal of Biochemistry

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Structural analysis of human profilin has revealed two tryptophan residues, W3 and W31, which interact with polyproline. The codons for these residues were mutated to encode phenylalanine and the mutant proteins overexpressed in Eschericia coli. The isolated proteins were diminished in their ability to bind polyproline, whereas phosphatidylinositol 4,5-bisphosphate (PIP 2 ) binding remained unchanged. In many strains of Saccharomyces cerevisiae, disruption of the gene encoding profilin, PFY1, is lethal. It was found that expression of the gene for human profilin is capable of suppressing this lethality. The polyproline-binding mutant alleles of the human gene were cloned into various yeast expression vectors. Each of the mutant genes resulted in suppression of the lethality of pfy1D. It was observed that the mutant protein expression levels paralleled the growth rates of the strains. The severity of various morphological abnormalities of the strains was also attenuated with increased protein levels, suggesting that profilin polyproline-binding mutations are deleterious to cell growth unless overexpressed. Both tryptophan mutations were combined to give a third mutant allele that was found both unable to bind polyproline and to suppress the lethality of a pfy1 deletion. Immunoprecipitation experiments suggested that the mutants were unaltered in their affinity for actin and PIP 2 . These data strongly suggest that polyproline binding is an essential function of profilin.Profilin is a ubiquitous eukaryotic protein that binds actin, phosphatidylinositol 4,5-bisphosphate (PIP 2 ) and poly-l-proline [1]. In resting mammalian cells, binding of profilin to PIP 2 inhibits phospholipid hydrolysis by phospholipase Cg (PLCg) [2]. When mammalian cells are stimulated to divide through the activation of receptor tyrosine kinases, PLCg becomes phosphorylated and hydrolyzes PIP 2 in the presence of profilin. It has been hypothesized that this hydrolysis not only releases the second messenger inositol trisphosphate from the plasma membrane but also releases profilin, allowing it to interact with actin in the cytosol [3]. When bound to actin, profilin has been shown to catalyze the release of ADP from monomeric (G-) actin [4], which increases the exchange of ADP for ATP. ATP/G-actin is activated for polymerization into microfilaments and facilitates the local reorganization of the cytoskeleton [5]. This competition between PIP 2 and actin for profilin binding is therefore an important regulatory component of the eukaryotic cell cycle. In the yeast Saccharomyces cerevisiae, the gene for profilin, PFY1, has been cloned and characterized [18]. Deletion of the gene is a lethal event in many strains [18,19], while in others, the disruption produces strains with highly abnormal morphologies and severely retarded growth rates [20]. The effects of yeast profilin on actin binding and polymerization have been studied and found to be very similar to those of multicellular organisms [21]. The PIP 2 -dependent translocation of profilin from t...

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Isolation and Characterization of theCandida albicans PFY1 Gene for Profilin

Cited by 4 publications

References 49 publications

Lack of Mitochondrial Anionic Phospholipids Causes an Inhibition of Translation of Protein Components of the Electron Transport Chain

Lack of Mitochondrial Anionic Phospholipids Causes an Inhibition of Translation of Protein Components of the Electron Transport Chain

Role of Actin Cytoskeletal Dynamics in Activation of the Cyclic AMP Pathway and HWP1 Gene Expression in Candida albicans

Polyproline binding is an essential function of human profilin in yeast

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