Ralf Egner

Isolation of autophagocytosis mutants of Saccharomyces cerevisiae

Protein degradation in the vacuole (lysosome) is an important event in cellular regulation. In yeast, as in mammalian cells, a major route of protein uptake for degradation into the vacuole (lysosome) has been found to be autophagocytosis. The discovery of this process in yeast enables the elucidation of its mechanisms via genetic and molecular biological investigations. Here we report the isolation of yeast mutants defective in autophagocytosis (aut mutants), using a rapid colony screening procedure.

The Pdr12 ABC transporter is required for the development of weak organic acid resistance in yeast

Exposure of Saccharomyces cerevisiae to sorbic acid strongly induces two plasma membrane proteins, one of which is identified in this study as the ATP‐binding cassette (ABC) transporter Pdr12. In the absence of weak acid stress, yeast cells grown at pH 7.0 express extremely low Pdr12 levels. However, sorbate treatment causes a dramatic induction of Pdr12 in the plasma membrane. Pdr12 is essential for the adaptation of yeast to growth under weak acid stress, since Δpdr12 mutants are hypersensitive at low pH to the food preservatives sorbic, benzoic and propionic acids, as well as high acetate levels. Moreover, active benzoate efflux is severely impaired in Δpdr12 cells. Hence, Pdr12 confers weak acid resistance by mediating energy‐dependent extrusion of water‐soluble carboxylate anions. The normal physiological function of Pdr12 is perhaps to protect against the potential toxicity of weak organic acids secreted by competitor organisms, acids that will accumulate to inhibitory levels in cells at low pH. This is the first demonstration that regulated expression of a eukaryotic ABC transporter mediates weak organic acid resistance development, the cause of widespread food spoilage by yeasts. The data also have important biotechnological implications, as they suggest that the inhibition of this transporter could be a strategy for preventing food spoilage.

The yeast multidrug transporter Pdr5 of the plasma membrane is ubiquitinated prior to endocytosis and degradation in the vacuole

We have recently demonstrated that the Pdr5 ATP binding cassette multidrug transporter is a short‐lived protein, whose biogenesis involves cell surface targeting followed by endocytosis and delivery to the vacuole for proteolytic turnover [Egner, R., Mahé, Y., Pandjaitan, R., and Kuchler, K. (1995) Mol. Cell. Biol. 15, 5879–5887]. Using c‐myc epitope‐tagged ubiquitin, we now have shown that Pdr5 is a ubiquitinated plasma membrane protein in vivo. Ubiquitination of Pdr5 was detected in both wild type and conditional end4 mutants defective in endocytic vesicle formation. Likewise, the Ste6 a‐factor pheromone transporter, which represents another short‐lived ABC transporter whose turnover requires vacuolar proteolysis, was also found to be ubiquitinated, and ubiquitin‐modified Ste6 massively accumulated in end4 mutants at the restrictive temperature. By contrast, the plasma membrane ATPase Pma1, a long‐lived and metabolically very stable protein, was found not to be ubiquitinated. Our results imply a novel function for ubiquitin in protein trafficking and suggest that ubiquitination of certain short‐lived plasma membrane proteins may trigger their endocytic delivery to the vacuole for proteolytic turnover.

Endocytosis and vacuolar degradation of the plasma membrane-localized Pdr5 ATP-binding cassette multidrug transporter in Saccharomyces cerevisiae.

Multidrug resistance (MDR) to different cytotoxic compounds in the yeast Saccharomyces cerevisiae can arise from overexpression of the Pdr5 (Sts1, Ydr1, or Lem1) ATP-binding cassette (ABC) multidrug transporter. We have raised polyclonal antibodies recognizing the yeast Pdr5 ABC transporter to study its biogenesis and to analyze the molecular mechanisms underlying MDR development. Subcellular fractionation and indirect immunofluorescence experiments showed that Pdr5 is localized in the plasma membrane. In addition, pulse-chase radiolabeling of cells and immunoprecipitation indicated that Pdr5 is a short-lived membrane protein with a half-life of about 60 to 90 min. A dramatic metabolic stabilization of Pdr5 was observed in delta pep4 mutant cells defective in vacuolar proteinases, and indirect immunofluorescence showed that Pdr5 accumulates in vacuoles of stationary-phase delta pep4 mutant cells, demonstrating that Pdr5 turnover requires vacuolar proteolysis. However, Pdr5 turnover does not require a functional proteasome, since the half-life of Pdr5 was unaffected in either pre1-1 or pre1-1 pre2-1 mutants defective in the multicatalytic cytoplasmic proteasome that is essential for cytoplasmic protein degradation. Immunofluorescence analysis revealed that vacuolar delivery of Pdr5 is blocked in conditional end4 endocytosis mutants at the restrictive temperature, showing that endocytosis delivers Pdr5 from the plasma membrane to the vacuole.

Reversal of antifungal resistance mediated by ABC efflux pumps from Candida albicans functionally expressed in yeast

The enhanced efflux of antifungal drugs through ATP-binding cassette (ABC) transporters constitutes a major cause of clinical multidrug resistance (MDR). The inhibition of drug efflux pumps by specific compounds is considered to be a feasible strategy to overcome clinical antifungal resistance. Therefore, several blockers of mammalian and yeast ABC drug pumps, including FK506, propafenones, as well as the antifungal drug terbinafine were tested for their capacity to reverse CDR-mediated azole resistance in bakers yeast and in clinical isolates of Candida albicans. We have functionally expressed the C. albicans Cdr1p and Cdr2p transporters in hypersensitive Saccharomyces cerevisiae recipient strains lacking several endogenous ABC pumps. Cdr1p and Cdr2p were functional in yeast, as they conferred pronounced drug resistance to known antifungal drugs, including azoles and terbinafine. We employ two functional assays to demonstrate that ABC pump inhibitors reverse CDR-mediated antifungal resistance, thereby restoring drug susceptibility of yeast cells and resistant clinical isolates. Our results suggest that reversal of antifungal resistance can be achieved through ABC pump-dependent and independent mechanisms.

The transmembrane domain 10 of the yeast Pdr5p ABC antifungal efflux pump determines both substrate specificity and inhibitor susceptibility.

We have previously shown that a S1360F mutation in transmembrane domain 10 (TMD10) of the Pdr5p ABC transporter modulates substrate specificity and simultaneously leads to a loss of FK506 inhibition. In this study, we have constructed and characterized the S1360F/A/T and T1364F/A/S mutations located in the hydrophilic face of the amphipatic Pdr5p TMD10. A T1364F mutation leads to a reduction in Pdr5p‐mediated azole and rhodamine 6G resistance. Like S1360F, the T1364F and T1364A mutants were nearly non‐responsive to FK506 inhibition. Most remarkably, however, the S1360A mutation increases FK506 inhibitor susceptibility, because Pdr5p–S1360A is hypersensitive to FK506 inhibition when compared with either wild‐type Pdr5p or the non‐responsive S1360F variant. Hence, the Pdr5p TMD10 determines both azole substrate specificity and susceptibility to reversal agents. This is the first demonstration of a eukaryotic ABC transporter where a single residue change causes either a loss or a gain in inhibitor susceptibility, depending on the nature of the mutational change. These results have important implications for the design of efficient reversal agents that could be used to overcome multidrug resistance mediated by ABC transporter overexpression.

ATP binding cassette transporters in yeast: From mating to multidrug resistance

Publisher Summary ATP binding cassette (ABC) transporters comprise a novel superfamily of membrane transport proteins that are found operating from bacteria to humans. More than 150 ABC sequences are deposited in the National Center for Biotechnology Information (NCBI) databank and their number keeps growing rapidly. The discovery that the Ste6 a-factor pheromone transporter represents a close homolog of the mammalian multidrug resistance P-glycoproteins motivated many laboratories to search for other yeast genes that are functionally or structurally related to STE6 . The joint efforts of many laboratories to systematically sequence the yeast genome has led to the identification of additional novel yeast ABC proteins. Therefore, the functional diversity of bacterial ABC transporters implies that even more ABC proteins can be discovered in both yeast and in mammalian systems.

Unusual Protein Secretion and Translocation Pathways in Yeast: Implication of ABC Transporters

In eukaryotic cells, the molecular machinery responsible for protein translocation across the endoplasmic reticulum (ER) membrane is well known. It comprises the cytoplasmic signal recognition particle, membrane receptors, chaperones and processing enzymes in the ER lumen.1 Peptides and proteins destined for secretion or which have to be transported to other intracellular organelles such as the lysosome are usually synthesized as larger precursors containing a cleavable signal peptide at or near the N-terminus of the polypeptide.2 Once properly assembled in the ER lumen, proteins are delivered to their final destinations through the exocytic pathway.3 Protein sorting and targeting is accomplished by vectorial generation of highly specialized transport vesicles in the ER and Golgi of both yeast4 and mammalian cells.3 The yeast Golgi complex also represents the cellular intersection where the proper sorting of plasma membrane proteins from vacuolar polypeptides is achieved.5,6

The Molecular Basis for Pleiotropic Drug Resistance in the Yeast Saccharomyces Cerevisiae: Regulation of Expression, Intracellular Trafficking and Proteolytic Turnover of ATP Binding Cassette (ABC) Multidrug Resistance Transporters

The major research goal of our laboratory is to understand the function mechanism of certain eukaryotic ABC transporters, including those from yeast as well as selected mammalian ABC proteins of medical importance. ABC transporters comprise the largest membrane transport protein family known to date, as more than 200 different ABC transporters have been identified operating from bacteria to man (Higgins, 1992; Kuchler and Thorner, 1992). The hallmark characteristics of all ABC-proteins include the presence of two highly conserved domains for ATP-binding (ABC), and two membrane domains each containing several predicted membrane-spanning α-helices (TMS). These four domains are normally arranged in an (TMS-ABC)2 or (ABC-TMS)2 configuration, although “half-size” transporters with an TMS-ABC or ABC-TMS topology and other topologies are also frequently found (Figure 1).