Daniel Kornitzer

Modes of Regulation of Ubiquitin-Mediated Protein Degradation

The ubiquitin‐proteasome pathway is responsible for the major portion of specific cellular protein degradation. Ubiquitin‐mediated degradation is involved in physiological regulation of many cellular processes, including cell cycle progression, differentiation, and signal transduction. Here, we review the basic mechanisms of the ubiquitin system and the various ways in which ubiquitin‐mediated degradation can be modulated by physiological signals. J. Cell. Physiol. 182:1–11, 2000.

Regulated degradation of the transcription factor Gcn4.

We report that Gcn4, a yeast transcriptional activator of the bZIP family involved in the regulation of the biosynthesis of amino acids and purines, is rapidly turned over. This degradation is inhibited under conditions of starvation for amino acids. Degradation is also inhibited by single amino acid alterations in a region adjacent to the Gcn4 activation domain. Furthermore, we show that degradation of Gcn4 proceeds through the ubiquitin pathway, a major proteolytic system for cytoplasmic proteins, and is dependent on two specific ubiquitin conjugating enzymes, Cdc34 (Ubc3) and Rad6 (Ubc2). As a first step towards reconstituting the Gcn4 degradation pathway in vitro, we show that purified Cdc34 and Rad6 proteins are able to direct the specific ubiquitination of Gcn4.

A family of Candida cell surface haem-binding proteins involved in haemin and haemoglobin-iron utilization

The ability to acquire iron from host tissues is a major virulence factor of pathogenic microorganisms. Candida albicans is an important fungal pathogen, responsible for an increasing proportion of systemic infections. C. albicans, like many pathogenic bacteria, is able to utilize haemin and haemoglobin as iron sources. However, the molecular basis of this pathway in pathogenic fungi is unknown. Here, we identify a conserved family of plasma membrane‐anchored proteins as haem‐binding proteins that are involved in haem‐iron utilization. We isolated RBT51 as a gene that is sufficient by itself to confer to S. cerevisiae the ability to utilize haemoglobin iron. RBT51 is highly homologous to RBT5, which was previously identified as a gene negatively regulated by the transcriptional suppressor CaTup1. Rbt5 and Rbt51 are mannosylated proteins that carry the conserved CFEM domain. We find that RBT5 is strongly induced by starvation for iron, and that deletion of RBT5 is by itself sufficient to significantly reduce the ability of C. albicans to utilize haemin and haemoglobin as iron sources. Iron starvation‐inducible, antigenically cross‐reacting haem‐binding proteins are also present in other Candida species that are able to utilize haem‐iron, underscoring the conservation of this iron acquisition pathway among pathogenic fungi.

Adenovirus E4orf4 protein induces PP2A-dependent growth arrest in Saccharomyces cerevisiae and interacts with the anaphase-promoting complex/cyclosome

Adenovirus early region 4 open reading frame 4 (E4orf4) protein has been reported to induce p53-independent, protein phosphatase 2A (PP2A)–dependent apoptosis in transformed mammalian cells. In this report, we show that E4orf4 induces an irreversible growth arrest in Saccharomyces cerevisiae at the G2/M phase of the cell cycle. Growth inhibition requires the presence of yeast PP2A-Cdc55, and is accompanied by accumulation of reactive oxygen species. E4orf4 expression is synthetically lethal with mutants defective in mitosis, including Cdc28/Cdk1 and anaphase-promoting complex/cyclosome (APC/C) mutants. Although APC/C activity is inhibited in the presence of E4orf4, Cdc28/Cdk1 is activated and partially counteracts the E4orf4-induced cell cycle arrest. The E4orf4–PP2A complex physically interacts with the APC/C, suggesting that E4orf4 functions by directly targeting PP2A to the APC/C, thereby leading to its inactivation. Finally, we show that E4orf4 can induce G2/M arrest in mammalian cells before apoptosis, indicating that E4orf4-induced events in yeast and mammalian cells are highly conserved.

An endocytic mechanism for haemoglobin‐iron acquisition in Candida albicans

The fungal pathogen Candida albicans is able to utilize haemin and haemoglobin as iron sources. Haem‐iron utilization is facilitated by Rbt5, an extracellular, glycosylphophatidylinositol (GPI)‐anchored, haemin‐ and haemoglobin‐binding protein. Here, we show that Rbt5 and its close homologue Rbt51 are short‐lived plasma membrane proteins, degradation of which depends on vacuolar activity. Rbt5 facilitates the rapid endocytosis of haemoglobin into the C.  albicans vacuole. We relied on recapitulation of the Rbt51‐dependent haem‐iron utilization in Saccharomyces cerevisiae to identify mutants defective in haemoglobin utilization. Homologues of representative mutants in S. cerevisiae were deleted in C. albicans and tested for haemoglobin‐iron utilization and haemoglobin uptake. These mutants define a novel endocytosis‐mediated haemoglobin utilization mechanism that depends on acidification of the lumen of the late secretory pathway, on a type I myosin and on the activity of the ESCRT pathway.

Alternative mRNA structures of the cIII gene of bacteriophage λ determine the rate of its translation initiation

The bacteriophage lambda cIII gene product has a regulatory function in the lysis-lysogeny decision following infection. The availability of a set of cIII expression mutants allowed us to establish the structure-function relationship of the cIII mRNA. We demonstrate, using defined in vitro systems, that the cIII mRNA is present in two conformations at equilibrium. Mutations that have been shown to lead to cIII overexpression were found to freeze the RNA in one conformation (structure B), and permit efficient binding to the 30 S ribosomal subunit. Mutations that have been shown to prevent cIII translation cause the mRNA to assume the alternative conformation (structure A). In this structure, the translation initiation region is occluded, thereby preventing 30 S ribosomal subunit binding. By varying the temperature or Mg2+ concentration it was possible to alter the relative proportion of the alternative structures in wild-type mRNA. We suggest that the regulation of the equilibrium between the two mRNA conformations provides a mechanism for the control of cIII gene expression.

Regulation of the transcription factor Gcn4 by Pho85 cyclin PCL5.

ABSTRACT The yeast transcription factor Gcn4 is regulated by amino acid starvation at the levels of both protein synthesis and stability. Gcn4 degradation depends on the ubiquitination complex SCFCDC4 and requires phosphorylation by the cyclin-dependent kinase Pho85. Here, we show that Pcl5 is the Pho85 cyclin specifically required for Gcn4 degradation. PCL5 is itself induced by Gcn4 at the level of transcription. However, even when PCL5 is constitutively overexpressed, Pho85-associated Gcn4 phosphorylation activity is reduced in starved cells and Gcn4 degradation is decreased. Under these conditions, the Pcl5 protein disappears because of rapid constitutive turnover. We suggest that, by virtue of its constitutive metabolic instability, Pcl5 may be a sensor of cellular protein biosynthetic capacity. The fact that PCL5 is transcriptionally induced in the presence of Gcn4 suggests that it is part of a homeostatic mechanism that reduces Gcn4 levels upon recovery from starvation.

Deletion of the copper transporter CaCCC2 reveals two distinct pathways for iron acquisition in Candida albicans

Efficient iron acquisition is an essential requirement for growth of pathogenic organisms in the iron‐poor host environment. In Saccharomyces cerevisiae, high‐affinity iron import depends on the multicopper ferroxidase ScFet3. ScFet3 biogenesis in the trans‐Golgi compartment requires a copper‐transporting P‐type ATPase, ScCcc2. Here, we describe the isolation by functional complementation of a Ccc2 homologue from the pathogenic yeast Candida albicans. CaCcc2 is functionally distinct from a previously described C. albicans copper‐transporting P‐type ATPase, CaCrp1, which appears to be specifically involved in copper detoxification. Regulation of CaCCC2 and the phenotype of the homozygous CaCCC2 deletion indicate that it is required for high‐affinity iron import, making it the bona fide CCC2 homologue of C. albicans. Remarkably, in a mouse model of systemic infection, the Caccc2Δ strain displayed robust proliferation and no significant reduction in pathogenicity, suggesting the existence of alternative mechanisms of iron uptake from host tissues. We identify haemin and haemoglobin as potential iron sources that can be used by C. albicans in a CaCcc2‐independent manner.

Novel small-molecule inhibitors of RNA polymerase III

ABSTRACT A genetic approach utilizing the yeast Saccharomyces cerevisiae was used to identify the target of antifungal compounds. This analysis led to the identification of small molecule inhibitors of RNA polymerase (Pol) III from Saccharomyces cerevisiae. Three lines of evidence show that UK-118005 inhibits cell growth by targeting RNA Pol III in yeast. First, a dominant mutation in the g domain of Rpo31p, the largest subunit of RNA Pol III, confers resistance to the compound. Second, UK-118005 rapidly inhibits tRNA synthesis in wild-type cells but not in UK-118005 resistant mutants. Third, in biochemical assays, UK-118005 inhibits tRNA gene transcription in vitro by the wild-type but not the mutant Pol III enzyme. By testing analogs of UK-118005 in a template-specific RNA Pol III transcription assay, an inhibitor with significantly higher potency, ML-60218, was identified. Further examination showed that both compounds are broad-spectrum inhibitors, displaying activity against RNA Pol III transcription systems derived from Candida albicans and human cells. The identification of these inhibitors demonstrates that RNA Pol III can be targeted by small synthetic molecules.

A Relay Network of Extracellular Heme-Binding Proteins Drives C. albicans Iron Acquisition from Hemoglobin

Iron scavenging constitutes a crucial challenge for survival of pathogenic microorganisms in the iron-poor host environment. Candida albicans, like many microbial pathogens, is able to utilize iron from hemoglobin, the largest iron pool in the hosts body. Rbt5 is an extracellular glycosylphosphatidylinositol (GPI)-anchored heme-binding protein of the CFEM family that facilitates heme-iron uptake by an unknown mechanism. Here, we characterize an additional C. albicans CFEM protein gene, PGA7, deletion of which elicits a more severe heme-iron utilization phenotype than deletion of RBT5. The virulence of the pga7−/− mutant is reduced in a mouse model of systemic infection, consistent with a requirement for heme-iron utilization for C. albicans pathogenicity. The Pga7 and Rbt5 proteins exhibit distinct cell wall attachment, and discrete localization within the cell envelope, with Rbt5 being more exposed than Pga7. Both proteins are shown here to efficiently extract heme from hemoglobin. Surprisingly, while Pga7 has a higher affinity for heme in vitro, we find that heme transfer can occur bi-directionally between Pga7 and Rbt5, supporting a model in which they cooperate in a heme-acquisition relay. Together, our data delineate the roles of Pga7 and Rbt5 in a cell surface protein network that transfers heme from extracellular hemoglobin to the endocytic pathway, and provide a paradigm for how receptors embedded in the cell wall matrix can mediate nutrient uptake across the fungal cell envelope.