John Lenard

CHARACTERIZATION OF PDR4, A SACCHAROMYCES CEREVISIAE GENE THAT CONFERS PLEIOTROPIC DRUG RESISTANCE IN HIGH-COPY NUMBER

PDR4 is a gene that confers pleiotropic drug resistance (pdr) to the yeast Saccharomyces cerevisiae when present in high copy number [Leppert et al., Genetics 125 (1990) 13-20]. Transposon insertion mutations had identified the active region of the gene as a 3.7-kb SalI-EcoRI restriction fragment of the 8-kb cloned fragment. We have confirmed this by showing that this fragment is sufficient to confer pdr, and have sequenced its entire 3761 bp. It contains a single complete open reading frame (ORF) extending from nucleotide (nt) position 1631-3580, coding for a protein of 650 amino acids (aa). A 2.7-kb fragment containing this ORF is also sufficient to confer pdr. The aa sequence contains no recognizable homologies or consensus sequences, so it is a novel protein of unknown function. It is apparently soluble, since no transmembrane-type sequences were predicted. A second, partial ORF was also found, on the opposite strand, extending from nt position 774 to past the SalI site, which is apparently unrelated to pdr.

Effects of growth state and amines on cytoplasmic and vocuolar pH, phosphate and polyphosphate levels in Saccharomyces cerevisiae: a 31P-nuclear magnetic resonance study

The vacuoles of logarithmic and stationary stage cells were compared by 31P-NMR with regard to pH, orthophosphate (Pi) content and average size of polyphosphate. The vacuoles of stationary cells had lower pH, higher Pi content, and polyphosphates of longer average chain length, although total polyphosphate content was about the same as in logarithmic cells. The lower vacuolar pH in stationary cells was the major cause of a larger cytoplasmic-vacuolar pH gradient. Addition of NH4Cl, (NH4)2SO4, methylamine or amantadine at pH 8 to cells in either stage caused an increase in both cytoplasmic and vacuolar pH, with little or no change in the cytoplasmic-vacuolar pH gradient. However, the administration of ammonium salts to the cells at pH 8.0 resulted in rapid hydrolysis of the intravacuolar polyphosphate to tripolyphosphate and Pi, with attendant redistribution of Pi between the vacuolar and cytoplasmic compartments.

Photoinactivation of influenza virus fusion and infectivity by rose bengal.

Rose bengal inactivated influenza virus upon exposure to light. Infectivity and fusion were inactivated with the same dose dependence, supporting the suggestion that the virucidal activity of photodynamic agents against enveloped viruses may be generally due to inactivation of their fusion protein(s). Concentrations required for inac‐ti vation were found to depend upon the ratio of rose bengal to virus, rather than on the nominal aqueous concentration. Fusion‐competent virosomes were inactivated similarly to intact virus particles. The HAZ portion of the influenza fusion protein HA underwent two different, apparently mutually exclusive modifications upon illumination with rose bengal: cross‐linking, and conversion to a form that moved slightly more slowly on sodium dodecyl sulfate poly‐acrylamide gel electrophoresis. Inactivation of viral fusion was inhibited by oxygen removal or addition of azide or β‐carotene, and was enhanced by D2O, consistent with partial involvement of singlet oxygen. The possibility of a second mechanism of viral photoinactivation, by direct interaction between the viral fusion protein and the pho‐toactivated dye, is also discussed.

Characterization of PDR4, a Saccharomyces cerevisiae gene that confers pleiotropic drug resistance in high-copy number: identity with YAP1, encoding a transcriptional activator

Abstract PDR4 is a gene that confers pleiotropic drug resistance (pdr) to the yeast Saccharomyces cerevisiae when present in high copy number [Leppert et al., Genetics 125 (1990) 13–20]. Transposon insertion mutations had identified the active region of the gene as a 3.7-kb Sal I- Eco RI restriction fragment of the 8-kb cloned fragment. We have confirmed this by showing that this fragment is sufficient to confer pdr, and have sequenced its entire 3761 bp. It contains a single complete open reading frame (ORF) extending from nucleotide (nt) position 1631–3580, coding for a protein of 650 amino acids (aa). A 2.7-kb fragment containing this ORF is also sufficient to confer pdr. The aa sequence contains no recognizable homologies or consensus sequences, so it is a novel protein of unknown function. It is apparently soluble, since no transmembrane-type sequences were predicted. A second, partial ORF was also found, on the opposite strand, extending from nt position 774 to past the Sal I site, which is apparently unrelated to pdr. [Just before receiving proofs, it was learned that PDR 4 was identical with YAP 1, a gene of the jun family encoding a transcriptional activator (see note added in proof).]

Radiation inactivation analysis of fusion and hemolysis by vesicular stomatitis virus

Radiation inactivation analysis was used to determine the size of the functional unit responsible for fusion of vesicular stomatitis virus (VSV) with cardiolipin or phosphatidylcholine-phosphatidylethanolamine (1:1) liposomes, and for VSV-induced hemolysis. When radiation-insensitive background values were subtracted, the calculated functional units for all three activities were similar, ranging from 866 to 957 kDa, equivalent to about 15 G protein molecules. This is in striking contrast to results of similar studies with influenza and Sendai viruses, in which the functional unit corresponded in size to a single fusion protein monomer, and suggests that VSV fusion may occur by a different mechanism.

A RELATIONSHIP BETWEEN MULTIDRUG RESISTANCE AND GROWTH-STATE DEPENDENT CYTOTOXICITY OF THE LYSOSOMOTROPIC DETERGENT N-DODECYLIMIDAZOLE

Multidrug resistance (MDR) in cultured cells and tumors is associated with overproduction of P-glycoprotein, a plasma membrane efflux pump normally present at very low levels. The cytotoxic action of N-dodecylimidazole (C12-Im), a lysosomotropic detergent, on cultured cells was previously shown to be strongly dependent on growth state, with rapidly growing cells being most sensitive and confluent cells most resistant. We show here that this may be due to a growth dependent increase in cellular P-glycoprotein activity. Both verapamil and nifedipine, structurally unrelated P-glycoprotein inhibitors, increased markedly the sensitivity of CHO fibroblasts to killing by C12-Im; the increase was greater in confluent than in growing cells. Also, verapamil inhibitable 3H-daunomycin efflux was more efficient from confluent than from subconfluent cells. The MDR cell line CH(R)C5 differed from all cell lines previously examined in that it did not show a growth-dependent decrease in C12-Im sensitivity, and sensitivity was not increased by verapamil or nifedipine. We suggest that a growth-dependent increase in MDR activity is a general property of cultured cells, except for those specifically overexpressing P-glycoprotein.

Fusion of a Sendai mutant deficient in HN protein (ts271) with cardiolipin liposomes.

Sendai mutant ts271 contains less than 5% of the amount of HN glycoprotein found in wild-type Sendai. Fusion of this mutant with cardiolipin liposomes revealed no differences from the wild-type virus with regard to specific activity, pH dependence, or radiation inactivation. Target sizes of both mutant and wild-type viral proteins were determined by the radiation-induced disappearance of each band from an SDS-polyacrylamide gel and no differences were found. Of the viral proteins, only F had a target size corresponding to the monomer molecular weight, ca. 60 kDa, identical to the minimum unit previously determined by functional assay for Sendai virus-erythrocyte membrane fusion (K. Bundo-Morita, S. Gibson, and J. Lenard, Biochemistry 26, 6223-6227 (1987)). This provides additional evidence that F alone is the active protein mediating Sendai-erythrocyte fusion. It is concluded that the HN protein is unlikely to mediate any fusion reactions of the intact virions, either with biological membranes or with cardiolipin liposomes.