WW domains of Rsp5p define different functions: determination of roles in fluid phase and uracil permease endocytosis in Saccharomyces cerevisiae.

Rsp5p, ubiquitin-protein ligase, an enzyme of the ubiquitination pathway, contains three WW domains that mediate protein-protein interactions. To determine if these domains adapt Rsp5p to a subset of substrates involved in numerous cellular processes, we generated mutations in individual or combinations of the WW domains. The rsp5-w1, rsp5-w2, and rsp5-w3 mutant alleles complement RSP5 deletions at 30 degrees. Thus, individual WW domains are not essential. Each rsp5-w mutation caused temperature-sensitive growth. Among variants with mutations in multiple WW domains, only rsp5-w1w2 complemented the deletion. Thus, the WW3 domain is sufficient for Rsp5p essential functions. To determine whether rsp5-w mutations affect endocytosis, fluid phase and uracil permease (Fur4p) endocytosis was examined. The WW3 domain is important for both processes. WW2 appears not to be important for fluid phase endocytosis whereas it is important for Fur4p endocytosis. In contrast, the WW1 domain affects fluid phase endocytosis, but it does not appear to function in Fur4p endocytosis. Thus, various WW domains play different roles in the endocytosis of these two substrates. Rsp5p is located in the cytoplasm in a punctate pattern that does not change during the cell cycle. Altering WW domains does not change the location of Rsp5p.

Suppressors of translation initiation defect in hem12 locus of Saccharomyces cerevisiae.

A system for the positive selection of transational initiation suppressors in S. cerevisiae has been developed. A mutant with an ATA initiation codon in the HEM12 gene, encoding uroporphyrinogen decarboxylase, was used to select cis- and trans-acting suppressors. These suppressors partially restore growth on nonfermentable carbon sources, such as glycerol, but still allow the accumulation of porphyrins. All extragenic suppressors are mapped to the SUI1 locus, encoding initiation factor eIF1. The effect of the hem12 mutation is also partially reversed by the known SUI3 suppressor encoding the beta subunit of eIF2. In contrast, the sui2 suppressor encoding the a subunit of eIF2 does not affect the hem12 phenotype. The intragenic suppressors are able to restore the translation of hem12 due to the generation of additional, in frame AUG codons upstream of the hem12-14 mutation. Mutational analysis of the HEM12 leader sequence was also performed to determine the role of small open reading frames (uORFs) present upstream of the HEM12 ORF. Studies on the expression of integrated hem12-1/4-lacZ fusion, devoid of all upstream ATGs, indicate a lack of regulatory effect of uORFs on HEM12 translation.

The growth of mdp1/rsp5 mutants of Saccharomyces cerevisiae is affected by mutations in the ATP-binding domain of the plasma membrane H+ -ATPase.

Mutations in the PMA1 gene, encoding plasma membrane H+ -ATPase, were isolated that are able to suppress the temperature sensitivity (ts) phenotype of mdp1 mutations located in RSP5, the ubiquitin-protein ligase gene. The mdp1 mutants were previously found to change the mitochondrial/cytosolic distribution of Mod5p-I, the tRNA modifying enzyme, and to affect fluid phase endocytosis. The data presented reveal that mdp1 mutants are also pH sensitive, and hypersensitive to hygromycin B and paromomycin. The ts phenotype, hygromycin B and paromomycin sensitivity are suppressed by pmal-t, but the pH sensitivity, the effect of mdp1 on Mod5p-I cytoplasmic/mitochondrial localization and endocytosis are not. Characterization of pmal-t revealed the substitution of amino acid G(653)V in the ATP-binding domain of the H+ -ATPase. Our results indicate that Rsp5 ubiquitin-protein ligase may also influence, in addition to protein distribution, the functioning of plasma membrane H+ -ATPase and the response of cells to stress.

Diamino acid derivatives of porphyrins penetrate into yeast cells, induce photodamage, but have no mutagenic effect.

The yeast Saccharomyces cerevisiae was used as a model eukaryotic organism to study the uptake of diamino acid derivatives of porphyrins and their phototoxicity with particular emphasis on possible mutagenic effects. The water-soluble hematoporphyrin derivatives diarginate (HpD[Arg]2) and 1-arginin di(N-amino acid)-protoporphyrinate used in this study are effective photosensitizers in tumor photodynamic therapy. Depending on the amino acid substituent, the porphyrin derivatives differ in their affinity for yeast cells. It is shown that HpD(Arg)2 and PP(Met)2 (Arg)2 penetrate into the yeast cell and are metabolized. Both compounds sensitize yeast cells to photodamage but have no mutagenic effect on nuclear or mitochondrial genomes.

MDP1, a Saccharomyces cerevisiae gene involved in mitochondrial/cytoplasmic protein distribution, is identical to the ubiquitin-protein ligase gene RSP5.

Alteration of the subcellular distribution of Mod5p-I, a tRNA modification enzyme, member of the sorting isozyme family, affects tRNA-mediated nonsense suppression. Altered suppression efficiency was used to identify MDP genes, which, when mutant, change the mitochondrial/cytosolic distribution of Mod5p-I,KR6. MDP2 is the previously identified VRP1, which encodes verprolin, required for proper organization of the actin cytoskeleton. MDP3 is identical to PAN1, which encodes a protein involved in initiation of translation and actin cytoskeleton organization. We report here the cloning and characterization of wild-type and mutant MDP1 alleles and the isolation and characterization of a multicopy suppressor of mdp1 mutations. MDP1 is identical to RSP5, which encodes ubiquitin-protein ligase, and mdp1 mutations are suppressed by high copy expression of ubiquitin. All four characterized mdp1 mutations cause missense changes located in the hect domain of Rsp5p that is highly conserved among ubiquitin-protein ligases. In addition to its well-known function in protein turnover, ubiquitination has been proposed to play roles in subcellular sorting of proteins via endocytosis and in delivery of proteins to peroxisomes, the endoplasmic reticulum and mitochondria. mdp1, as well as mdp2/vrp1 and mdp3/pan1 mutations, affect endocytosis. Further, mdp1 mutations show synthetic interactions with mdp2/vrp1 and mdp3/pan1. Identification of MDP1 as RSP5, along with our previous identification of MDP2/VRP1 and MDP3/PAN1, implicate interactions of the ubiquitin system, the actin cytoskeleton and protein synthesis in the subcellular distribution of proteins.

Mutation in a new gene MAF1 affects tRNA suppressor efficiency in Saccharomyces cerevisiae.

Mutation in the MAF1 gene was identified in a screen for decreased efficiency of tRNA suppressor SUP11 in the yeast Saccharomyces cerevisiae (Sc). maf1-1 mutation exerts a dual phenotypic effect: antisuppression and temperature sensitive (ts) respiratory growth. MAF1, cloned by complementation of the ts phenotype of maf1-1, also alleviates the antisuppressor effect. The coding sequence of MAF1 is interrupted by an intron of 80 bp. The putative gene product, Maf1p, is a hydrophilic protein of 395 amino acids (aa) not showing significant similarity to known proteins which indicates that MAF1 encodes a novel protein. Maf1p may play a role in the tRNA biosynthetic pathway since a fragment of the RPO31/RPC160 gene encoding the largest subunit of RNA polymerase III was cloned as a multicopy suppressor of mafl-1.

Saccharomyces cerevisiae mutants defective in heme biosynthesis as a tool for studying the mechanism of phototoxicity of porphyrins.

Mutants of Saccharomyces cerevisiae accumulating uroporphyrin (UP) or protoporphyrin (PP) were used as a model for the in vivo phototoxic effect of porphyrins observed in the human skin photosensitivity associated with porphyrias (porphyria cutanea tarda and erythropoietic protoporphyria). We have found that UP is localized in vacuoles and PP is present in all compartments except vacuoles in yeast cells. Endogenous PP is much more effective as a photosensitizer of yeast cells than UP. Protoporphyrin action is strictly dependent on the presence of oxygen. In contrast, UP displays a phototoxic effect even if oxygen is not present in the suspension, implicating a free radical mechanism that operates in anaerobiosis upon photosensitization by UP. Catalase or superoxide dismutase deficiency affects photosensitization by UP. A possible mechanism of UP photosensitizing activity is discussed.

Mutations altering the mitochondrial-cytoplasmic distribution of Mod5p implicate the actin cytoskeleton and mRNA 3' ends and/or protein synthesis in mitochondrial delivery.

The Saccharomyces cerevisiae MOD5 gene encodes proteins that function in three subcellular locations: mitochondria, the cytoplasm, and nuclei (M. Boguta, L.A. Hunter, W.-C. Shen, E. C. Gillman, N. C. Martin, and A. K. Hopper, Mol. Cell. Biol. 14:2298-2306, 1994; E. C. Gillman, L. B. Slusher, N. C. Martin, and A. K. Hopper, Mol. Cell. Biol. 11:2382-2390, 1991). A mutant allele of MOD5 encoding a protein (Mod5p-I,KR6) located predominantly in mitochondria was constructed. Mutants defective in delivering Mod5p-I,KR6 to mitochondria were sought by selecting cells with increased cytosolic activity of this protein. Twenty-five mutants defining four complementation groups, mdp1, mdp2, mdp3, and mdp4, were found. They are unable to respire at 34 degrees C or to grow on glucose medium at 38 degrees C. Cell fractionation studies showed that mdp1, mdp2, and mdp3 mutants have an altered mitochondrial-cytoplasmic distribution of Mod5p. mdp2 can be suppressed by ACT1, the actin-encoding gene. The actin cytoskeleton organization is also aberrant in mdp2 cells. MDP2 is the same as VRP1 (S. F. H. Donnelly, M. J. Picklington, D. Pallotta, and E. Orr, Mol. Microbiol. 10:585-596, 1993). MDP3 is identical to PAN1, which encodes a protein that interacts with mRNA 3' ends and affects initiation of protein synthesis (A. B. Sachs and J. A. Deardoff, Cell 70:961-973, 1992). These results implicate the actin cytoskeleton and mRNA 3' ends and/or protein synthesis as being as important for protein distribution in S. cerevisiae as they are for distribution of cytosolic proteins in higher eukaryotes. This provides the potential to apply genetic and molecular approaches to study gene products and mechanisms involved in this type of protein distribution. The selection strategy also offers a new approach for identifying gene products involved in the distribution of proteins to their subscellular destinations.

Isolation and characterization of extragenic mutations affecting the expression of the uroporphyrinogen decarboxylase gene (HEM12) in Sacharomyces cerevisiae.

Uroporphyrinogen decarboxylase (Uro-d; EC 4.1.1.37), the fifth enzyme in the heme biosynthetic pathway, which catalyzes the sequential decarboxylation of uroporphyrinogen to coproporphyrinogen, is encoded by the HEM12 gene in Saccharomyces cerevisiae. The HEM12 gene is transcribed into a major short mRNA and a minor longer one, approximately 1.35 and 1.55 kb, respectively, in size, and that differ in the 5' untranslated region. "Uroporphyric" mutants, which have no mutations in the HEM12 gene but accumulate uroporphyrinogen, a phenotype characteristic of partial Uro-d deficiency, were investigated. Genetic analysis showed that the mutant phenotype depends on the combined action of two unlinked mutations, udt1 and either ipa1, ipa2, or ipa3. ipa1 is tightly linked to HEM12. The mutation udt1 apparently acts specifically on the HEM12 gene, and causes a six to tenfold decrease in the levels of the short HEM12 mRNA, in the beta-galactosidase activity of a HEM12-lacZ fusion, in immunodetectable protein and enzyme activity. But heme synthesis is normal and porphyrin accumulation was modest. The mutations ipa1, ipa2, and ipa3 had no phenotype on their own, but they caused an increase in porphyrin accumulation in a udt1 background. This multiplicity of genetic factors leading to uroporphyric yeast cells closely resembles the situation in human porphyria cutanea tarda.

maf1 mutation alters the subcellular localization of the Mod5 protein in yeast.

Two forms of Mod5p, a tRNA modification enzyme, are found in three intracellular compartments, mitochondria, cytoplasm and nucleus, but are encoded by a single MOD5 gene. The two forms of the enzyme, Mod5p-I and Mod5p-II differ at the N-termini and are produced by initiation of translation at different start codons. Mod5p-I does contain a mitochondrial targeting signal and is distributed between mitochondria and cytoplasm, whereas Mod5p-II is found in the cytosol and nucleus (Boguta, M., et al. 1994, Mol. Cell. Biol. 14, 2298-2306). In the present work mutants which mislocalize the Mod5p-I enzyme were isolated. The screen was based on a correlation between the amount of cytosolic protein and the efficiency of tRNA mediated suppression. Identification of mutants is possible because a red pigment accumulates in the cells unable to suppress an ade2-1 nonsense allele. The maf1 mutant, with an altered intracellular localization of the Mod5p-I protein, was isolated. Immunofluorescence data suggest that the mutation causes mislocalization of the Mod5p-I to the nucleus.

Identification of amino acid changes affecting yeast uroporphyrinogen decarboxylase activity by sequence analysis of hem12 mutant alleles.

The molecular basis of the uroporphyrinogen decarboxylase defect in eleven yeast 'uroporphyric' mutants was investigated. Uroporphyrinogen decarboxylase, an enzyme of the haem-biosynthetic pathway, catalyses the decarboxylation of uroporphyrinogen to coproporphyrinogen and is encoded by the HEM12 gene in the yeast Saccharomyces cerevisiae. The mutations were identified by sequencing the mutant hem12 alleles amplified in vitro from genomic DNA extracted from the mutant strains. Four mutations leading to the absence of enzyme protein were found: one mutation caused the substitution of the translation initiator Met to Ile, a two-base deletion created a frameshift at codon 247 and two nonsense mutations were found at codons 50 and 263. Four different point mutations were identified in seven 'leaky' mutants with residual modified uroporphyrinogen decarboxylase activity; each of three mutations was found in two independently isolated mutants. The nucleotide transitions resulted in the amino acid substitutions Ser-59 to Phe, Thr-62 to Ile, Leu-107 to Ser, or Ser-215 to Asn, all located in or near highly conserved regions. The results suggest that there is a single active centre in uroporphyrinogen decarboxylase, the geometry of which is affected in the mutant enzymes.

The effects in vivo of mutationally modified uroporphyrinogen decarboxylase in different hem12 mutants of baker's yeast (Saccharomyces cerevisiae).

Nine new hem12 haploid mutants of baker's yeast (Saccharomyces cerevisiae), totally or partially deficient in uroporphyrinogen decarboxylase activity, were subjected to both genetic and biochemical analysis. The mutations sites studied are situated far apart within the HEM12 gene located on chromosome IV. Uroporphyrinogen decarboxylase activity in the cell-free extracts of the mutants was decreased by 50-100%. This correlated well with the decrease of haem formation and the increased accumulation and excretion of porphyrins observed in vivo. The pattern of porphyrins (uroporphyrin and its decarboxylation products) accumulated in the cells of mutants partially deficient in uroporphyrinogen decarboxylase activity did not differ significantly, although differences in vitro were found in the relative activity of the mutant enzyme at the four decarboxylation steps. The excreted porphyrins comprised mainly dehydroisocoproporphyrin or pentacarboxyporphyrin. In heterozygous hem12-1/HEM12 diploid cells, a 50% decrease in decarboxylase activity led to an increased accumulation of porphyrins as compared with the wild-type HEM12/HEM12 diploid, which points to the semi-dominant character of the hem12-1 mutation. The biochemical phenotypes of both the haploid and the heterozygous diploid resembles closely the situation encountered in porphyria cutanea tarda, the most common human form of porphyria.

Nuclear suppressors of the mitochondrial mutation oxi1-V25 in Saccharomyces cerevisiae. Genetic analysis of the suppressors: absence of complementation between non-allelic mutants.

Ten informational nuclear suppressors of the oxi1- mitochondrial mutation of Saccharomyces cerevisiae are recessive. They are linked to each other, but their allelism is uncertain. Some of them unfavourably affect functions of standard (mit+) mitochondrial genomes. One suppressor severely impairs or entirely prevents mitochondrial functions of the spore clones carrying it. The spectrum of mit- mutations on which these suppressors act is similar to that exhibited by nam3-1. In double heterozygotes namx/NAM3+, NAM+x/nam3-1 the oxi1- (and box3-) mutation is suppressed, yet one of our suppressors (R705) and nam3-1 show independent segregation in tetrads. This indicates that there may be absence of complementation between non-allelic suppressors.