A novel EH domain protein of Saccharomyces cerevisiae, Ede1p, involved in endocytosis.

Sequencing of the entire genome of S. cerevisiae has revealed the existence of five proteins containing EH domains. These are protein-protein interaction modules first described in mammalian Eps15, a protein that is involved in clathrin-dependent endocytosis. Two of the yeast proteins, End3p and Pan1p, are required for the internalization step of endocytosis. We report characterization of the nonessential ORF YBL047c which, like Eps15, encodes a protein with three N-terminal EH domains. Deletion of YBL047c leads to a defective fluid-phase endocytosis and to defective internalization of the pheromone (alpha)-factor and uracil permease. We therefore named YBL047c EDE1, for EH Domains and Endocytosis. Ede1p expressed as a chromosomally encoded fusion to the green fluorescent protein is localized in punctate cortical spots that only partially colocalize with actin patches. This localization is maintained when actin is depolymerized. Deletion of EDE1 impairs the diploid budding pattern, but has only a small impact on actin cytoskeleton organization, in contrast to the effects observed in pan1 cells and many end mutants impaired in proteins colocalizing with cortical actin patches. Genetic interaction was observed between EDE1 and RSP5, which encodes the ubiquitin ligase Rsp5p essential for ubiquitin-dependent endocytosis of many plasma membrane proteins, thus further emphasizing the functional link between Rsp5p and the EH domain proteins. We also observed genetic interaction between EDE1, and END3 or PAN1, suggesting that Ede1p might be part of a yeast EH network implicated in endocytosis.

Identification of the genes encoding the cytosolic translation release factors from Podospora anserina and analysis of their role during the life cycle.

In an attempt to decipher their role in the life history and senescence process of the filamentous fungus Podospora anserina, we have cloned the su1 and su2 genes, previously identified as implicated in cytosolic translation fidelity. We show that these genes are the equivalents of the SUP35 and SUP45 genes of Saccharomyces cerevisiae, which encode the cytosolic translation termination factors eRF3 and eRF1, respectively. Mutations in these genes that suppress nonsense mutations may lead to drastic mycelium morphology changes and sexual impairment but have little effect on life span. Deletion of su1, coding for the P. anserina eRF3, is lethal. Diminution of its expression leads to a nonsense suppressor phenotype whereas its overexpression leads to an antisuppressor phenotype. P. anserina eRF3 presents an N-terminal region structurally related to the yeast eRF3 one. Deletion of the N-terminal region of P. anserina eRF3 does not cause any vegetative alteration; especially life span is not changed. However, it promotes a reproductive impairment. Contrary to what happens in S. cerevisiae, deletion of the N terminus of the protein promotes a nonsense suppressor phenotype. Genetic analysis suggests that this domain of eRF3 acts in P. anserina as a cis-activator of the C-terminal portion and is required for proper reproduction.

[Genetic analysis of two cellular degenerations in filamentous fungus Podospora anserina]. / Génétique de deux dégénérescences cellulaires chez le champignon filamenteux Podospora anserina.

The filamentous fungus Podopsora anserina presents an unavoidable arrest of vegetative growth (Senescence) determined by a cytoplasmic and infectious factor. Senescence is correlated with a disorganization of the mitochondrial DNA. This disorganization is caused by an event which is not the appearance of the first defective DNA molecules. These ones are generated constitutively and their accumulation during Senescence requires the presence of an additional factor. Life span of the strains is under nuclear and cytoplasmic genetic control. At least 600 nuclear genes influence longevity. Our analysis focuses on the role of the genes involved in cytosolic translation, since mutations in these genes seem to display the most drastic effects on longevity but also on the structure of the defective mitochondrial DNA molecules that accumulate during Senescence. We have detected in some Podospora anserina mutant strains (permissive strains) the presence of a novel cytoplasmic and infectious determinant that entails an easily discernible phenotype associated with a severe growth alteration (Crippled Growth). This growth alteration is not associated with mitochondrial DNA modifications. Only the strains that have an increased translational accuracy present Crippled Growth. However, the Crippled Growth Determinant is found in all the strains during the stationary phase; it is eliminated from the non permissive strains during the exit of the stationary phase. The mutants, that have an increased translational accuracy, probably lack a factor which is needed to eliminate the determinant when cells enter the growth phase.

Cloning, sequencing, and transgenic expression of Podospora curvicolla and Sordaria macrospora eEF1A genes: relationship between cytosolic translation and longevity in filamentous fungi.

We have cloned and sequenced the gene encoding the translation elongation factor eEF1A from two filamentous fungi, Podospora curvicolla and Sordaria macrospora. These fungi are close relatives of Podospora anserina and also show senescence syndromes. Comparison of the sequences of the deduced proteins with that of P. anserina reveals that the three proteins differ in several positions. Replacement of the P. anserina gene by either of the two exogenous genes does not entail any modification in P. anserina physiology; the longevity of the fungus is not affected. No alteration of in vivo translational accuracy was detected; however, the exogenous proteins nonetheless promoted a modification of the resistance to the aminoglycoside antibiotic paromomycin. These data suggest that optimization of life span between these closely related fungi has likely not been performed during evolution through modifications of eEF1A activity, despite the fact that mutations in this factor can drastically affect longevity.

Mecillinam resistance in Escherichia coli is conferred by loss of a second activity of the AroK protein.

Mecillinam, a beta-lactam antibiotic specific to penicillin-binding protein 2 (PBP 2) in Escherichia coli, blocks cell wall elongation and, indirectly, cell division, but its lethality can be overcome by increased levels of ppGpp, the nucleotide effector of the stringent response. We have subjected an E. coli K-12 strain to random insertional mutagenesis with a mini-Tn10 element. One insertion, which was found to confer resistance to mecillinam in relA+ and relA strains, was mapped at 75.5 min on the E. coli map and was located between the promoters and the coding sequence of the aroK gene, which codes for shikimate kinase 1, one of two E. coli shikimate kinases, both of which are involved in aromatic amino acid biosynthesis. The mecillinam resistance conferred by the insertion was abolished in a delta relA delta spoT strain completely lacking ppGpp, and it thus depends on the presence of ppGpp. Furthermore, the insertion increased the ppGpp pool approximately twofold in a relA+ strain. However, this increase was not observed in relA strains, although the insertion still conferred mecillinam resistance in these backgrounds, showing that mecillinam resistance is not due to an increased ppGpp pool. The resistance was also abolished in an ftsZ84(Ts) strain under semipermissive conditions, and the aroK::mini-Tn10 allele partially suppressed ftsZ84(Ts); however, it did not increase the concentration of the FtsZ cell division protein. The insertion greatly decreased or abolished the shikimate kinase activity of AroK in vivo and in vitro. The two shikimate kinases of E. coli are not equivalent; the loss of AroK confers mecillinam resistance, whereas the loss of Arol, does not. Furthermore, the ability of the aroK mutation to confer mecillinam resistance is shown to be independent of polar effects on operon expression and of effects on the availability of aromatic amino acids or shikimic acid. Instead, we conclude that the AroK protein has a second activity, possibly related to cell division regulation, which confers mecillinam sensitivity. We were able to separate the AroK activities mutationally with an aroK mutant allele lacking shikimate kinase activity but still able to confer mecillinam sensitivity.