T URNOVER OF AB ERR ANT PROT EINS IN E. COLI . . . . . . . . . . . . . . . . . 445 The Lon Protease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 446 DnaK. DnaJ. GrpE. GroEL and GroES . . . . . . . . . . . . . . . . . . . . . . . . . . 447 The Clp Protease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 448 The DegP Protease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 452

The Function of Heat-Shock Proteins in Stress Tolerance: Degradation and Reactivation of Damaged Proteins

In the past decade, the frequency of diagnosed fungal infections has risen sharply due to several factors, including the increase in the number of immunosuppressed patients resulting from the AIDS epidemic and treatments during and after organ and bone marrow transplants. Linked with the increase in fungal infections is a recent increase in the frequency with which these infections are recalcitrant to standard antifungal therapy. This review summarizes the factors that contribute to antifungal drug resistance on three levels: (i) clinical factors that result in the inability to successfully treat refractory disease; (ii) cellular factors associated with a resistant fungal strain; and (iii) molecular factors that are ultimately responsible for the resistance phenotype in the cell. Many of the clinical factors that contribute to resistance are associated with the immune status of the patient, with the pharmacology of the drugs, or with the degree or type of fungal infection present. At a cellular level, antifungal drug resistance can be the result of replacement of a susceptible strain with a more resistant strain or species or the alteration of an endogenous strain (by mutation or gene expression) to a resistant phenotype. The molecular mechanisms of resistance that have been identified to date in Candida albicans include overexpression of two types of efflux pumps, overexpression or mutation of the target enzyme, and alteration of other enzymes in the same biosynthetic pathway as the target enzyme. Since the study of antifungal drug resistance is relatively new, other factors that may also contribute to resistance are discussed.

Clinical, cellular, and molecular factors that contribute to antifungal drug resistance.

Foreign gene expression in yeast: a review.

Biochemistry of metallothionein

Interactions of toxic metals with fungi have long been of importance since metal toxicity still is the basis of many fungicidal preparations while in an environmental context, accelerating pollution of the natural environment has led to increased interest because of the sometimes dominant presence of fungi in metal-polluted habitats, the translocation of toxic metals and radionuclides to fruit bodies of edible higher fungi, and the significance of mycorrhizal fungi in the amelioration of metal phytotoxicity (Colpaert and Van Assche, 1987). Furthermore, the use of fungal (and other microbial) biomass for the detoxification of metal/radionuclide-containing industrial effluents is of biotechnological potential (Gadd, 1990, 1992a).

Interactions of fungip with toxic metals

Addition of copper to yeast cells leads to the induction of a low molecular weight, cysteine-rich protein that binds copper. This protein, termed copper chelatin or thionein, is related to the metallothionein family of proteins that are induced in response to cadmium and zinc in vertebrate cells. We have determined the structure of the yeast copper-binding protein by DNA sequence analysis of the gene. Although the 6573-dalton yeast protein is substantially divergent from vertebrate metallothioneins, the arrangement of 12 cysteine residues, which is a hallmark of metal-binding proteins, is partially conserved. We analyzed the regulatory DNA sequence of the gene by fusing it with the Escherichia coli galactokinase gene and assaying the levels of enzyme activity in yeast in response to copper. The transcriptional activation has a specific requirement for copper. Zinc, cadmium, and gold were unable to regulate the galactokinase activity. The yeast copper metallothionein regulatory sequences represent a previously unreported class of yeast promoter that is regulated by copper.

https://www.pnas.org/content/pnas/81/11/3332.full.pdf

Copper metallothionein of yeast, structure of the gene, and regulation of expression

The pathogenic yeast, Candida albicans, is insensitive to the anti-mitotic drug, benomyl, and to the dihydrofolate reductase inhibitor, methotrexate. Genes responsible for the intrinsic drug resistance were sought by transforming Saccharomyces cerevisiae, a yeast sensitive to both drugs, with genomic C. albicans libraries and screening on benomyl or methotrexate. Restriction analysis of plasmids isolated from benomyl- and methotrexate-resistant colonies indicated that both phenotypes were encoded by the same DNA fragment. Sequence analysis showed that the fragments were nearly identical and contained a long open reading frame of 1694 bp (ORF1) and a small ORF of 446 bp (ORF2) within ORF1 on the opposite strand. By site-directed mutagenesis, it was shown that ORF1 encoded both phenotypes. The protein had no sequence similarity to any known proteins, including β-tubulin, dihydrofolate reductase, and the P-glycoprotein of the multi-drug resistance family. The resistance gene was detected in several C. albicans strains and in C. stellatoidea by DNA hybridization and by the polymerase chain reaction.

Jessica A. Gorman

Papers

Copper metallothionein of yeast, structure of the gene, and regulation of expression

Analysis of a Candida albicans gene that encodes a novel mechanism for resistance to benomyl and methotrexate.

Yeast metallothionein function in metal ion detoxification.

Increasing gene expression in yeast by fusion to ubiquitin.

Isolation and characterization of a β-tubulin gene from Candida albicans