We have screened a collection of haploid yeast knockout strains for increased sensitivity to 5-fluorouracil (5-FU). A total of 138 5-FU sensitive strains were found. Mutants affecting rRNA and tRNA maturation were particularly sensitive to 5-FU, with the tRNA methylation mutant trm10 being the most sensitive mutant. This is intriguing since trm10, like many other tRNA modification mutants, lacks a phenotype under normal conditions. However, double mutants for nonessential tRNA modification enzymes are frequently temperature sensitive, due to destabilization of hypomodified tRNAs. We therefore tested if the sensitivity of our mutants to 5-FU is affected by the temperature. We found that the cytotoxic effect of 5-FU is strongly enhanced at 38°C for tRNA modification mutants. Furthermore, tRNA modification mutants show similar synthetic interactions for temperature sensitivity and sensitivity to 5-FU. A model is proposed for how 5-FU kills these mutants by reducing the number of tRNA modifications, thus destabilizing tRNA. Finally, we found that also wild-type cells are temperature sensitive at higher concentrations of 5-FU. This suggests that tRNA destabilization contributes to 5-FU cytotoxicity in wild-type cells and provides a possible explanation why hyperthermia can enhance the effect of 5-FU in cancer therapy.

Abstract. Phagosome-lysosome membrane fusion is a highly regulated event that is essential for intracellular killing of microorganisms. Functionally, it represents a form of polarized regulated secretion, which is classically dependent on increases in intracellular ionized calcium ([Ca2+]i). Indeed, increases in [Ca2+]i are essential for phagosome-granule (lysosome) fusion in neutrophils and for lysosomal fusion events that mediate host cell invasion by Trypanosoma cruzi trypomastigotes. Since several intracellular pathogens survive in macrophage phagosomes that do not fuse with lysosomes, we examined the regulation of phagosome-lysosome fusion in macrophages. Macrophages (MO) were treated with 12.5 IxM bis-(2-amino-S-methylphenoxy) ethane-N,N,N',N',-tetraacetic acid tetraacetoxymethylester (MAPT/AM), a cell-permeant calcium chelator which reduced resting cytoplasmic [Ca2+]; from 80 nM to ~<20 nM and completely blocked increases in [Ca2+]i in response to multiple stimuli, even in the presence of extracellular calcium. Subsequently, M~ phagocytosed serum-opsonized zymosan, staphylococci, or Mycobacterium bovis. Microbes were enumerated by 4',6-diamidino-2-phenylindole, dihydrochloride (DAPI) staining, and phagosome-lysosome fusion was scored using both lysosome-associated membrane protein (LAMP-l) as a membrane marker and rhodamine dextran as a content marker for lysosomes. Confirmation of phagosomelysosome fusion by electron microscopy validated the fluorescence microscopy findings. We found that phagosome-lysosome fusion in MO occurs normally at very low [Ca2+]i (~<20 nM). Kinetic analysis showed that in MO none of the steps leading from particle binding to eventual phagosome-lysosome fusion are regulated by [Ca2+]i in a rate-limiting way. Furthermore, confocal microscopy revealed no difference in the intensity of LAMP-1 immunofluorescence in phagolysosome membranes in calcium-buffered vs. control macrophages. We conclude that neither membrane recognition nor fusion events in the phagosomal pathway in macrophages are dependent on or regulated by calcium.AGOCYTOSIS is a tightly regulated process leading o the internalization of particulate ligands such as acteria and fungi. Its initiation depends on the engagement of specific plasma membrane receptors expressed on macrophages and neutrophils. In a manner analogous to that of hormones and growth factors, binding of particulate ligands to phagocytic receptors triggers intraceUular signals. One common intracellular signaling event that accompanies phagocytosis is an increase in in-

5-Fluorouracil (5-FU) is an anticancer drug and pyrimidine analogue. A problem in 5-FU therapy is acquired resistance to the drug. To find out more about the mechanisms of resistance, we screened a plasmid library in yeast for genes that confer 5-FU resistance when overexpressed. We cloned five genes: CPA1, CPA2, HMS1, HAM1 and YJL055W. CPA1 and CPA2 encode a carbamoyl phosphate synthase involved in arginine biosynthesis and HMS1 a helix-loop-helix transcription factor. Our results suggest that CPA1, CPA2, and HMS1 confer 5-FU resistance by stimulating pyrimidine biosynthesis. Thus, they are unable to confer 5-FU resistance in a ura2 mutant, and inhibit the uptake and incorporation into RNA of both uracil and 5-FU. In contrast, HAM1 and YJL055W confer 5-FU resistance in a ura2 mutant, and selectively inhibit incorporation into RNA of 5-FU but not uracil. HAM1 is the strongest resistance gene, but it partially depends on YJL055W for its function. This suggests that HAM1 and YJL055W function together in mediating resistance to 5-FU. Ham1p encodes an inosine triphosphate pyrophosphatase that has been implicated in resistance to purine analogues. Our results suggest that Ham1p could have a broader specificity that includes 5-FUTP and other pyrimidine analogoue triphosphates.

We have screened a complete collection of yeast knockout mutants for sensitivity to monensin, an ionophore that interferes with intracellular transport. A total of 63 sensitive strains were found. Most of the strains were deleted for genes involved in post-Golgi traffic, with an emphasis on vacuolar biogenesis. A high correlation was thus seen with VPS and VAM genes, but there were also significant differences between the three sets of genes. A weaker correlation was seen with sensitivity to NaCl, in particular rate of growth effects. Interestingly, all 14 genes encoding subunits of the vacuolar H(+)-ATPase (V-ATPase) were absent in our screen, even though they appeared in the VPS or VAM screens. All monensin-sensitive mutants that could be tested interact synthetically with a deletion of the A subunit of the V-ATPase, Vma1. Synthetic lethality was limited to mutations affecting endocytosis or retrograde transport to Golgi. In addition, vma1 was epistatic over the monensin sensitivity of vacuolar transport mutants, but not endocytosis mutants. Deletions of the two isoforms of the V-ATPase a subunit, Vph1 and Stv1 had opposite effects on the monensin sensitivity of a ypt7 mutant. These findings are consistent with a model where monensin inhibits growth by interfering with the maintenance of an acidic pH in the late secretory pathway. The synthetic lethality of vma1 with mutations affecting retrograde transport to the Golgi further suggests that it is in the late Golgi that a low pH must be maintained.