C173 and W125 are pet mutants of Saccharomyces cerevisiae, partially deficient in cytochrome oxidase but with elevated concentrations of cytochrome c. Assays of electron transport chain enzymes indicate that the mutations exert different effects on the terminal respiratory pathway, including an inefficient transfer of electrons between the bc 1 and the cytochrome oxidase complexes. A cloned gene capable of restoring respiration in C173/U1 and W125 is identical to reading frame YGR112w of yeast chromosome VII (GenBank Z72897). The encoded protein is homologous to the product of the mammalian SURF-1 gene. In view of the homology, the yeast gene has been designated SHY1 (Surf Homolog of Yeast). An antibody against the carboxyl-terminal half of Shy1p has been used to localize the protein in the inner mitochondrial membrane. Deletion of part of SHY1 produces a phenotype similar to that of G91 mutants. Disruption of SHY1 at a BamHI site, located approximately 2/3 of the way into the gene, has no obvious phenotypic consequence. This evidence, together with the ability of a carboxyl-terminal coding sequence starting from the BamHI site to complement a shy1 mutant, suggests that the Shy1p contains two domains that can be separately expressed to form a functional protein.

The FAD1 gene of Saccharomyces cerevisiae has been selected from a genomic library on the basis of its ability to partially correct the respiratory defect of pet mutants previously assigned to complementation group G178. Mutants in this group display a reduced level of flavin adenine dinucleotide (FAD) and an increased level of flavin mononucleotide (FMN) in mitochondria. The restoration of respiratory capability by FAD1 is shown to be due to extragenic suppression. FAD1 codes for an essential yeast protein, since disruption of the gene induces a lethal phenotype. The FAD1 product has been inferred to be yeast FAD synthetase, an enzyme that adenylates FMN to FAD. This conclusion is based on the following evidence. S. cerevisiae transformed with FAD1 on a multicopy plasmid displays an increase in FAD synthetase activity. This is also true when the gene is expressed in Escherichia coli. Lastly, the FAD1 product exhibits low but significant primary sequence similarity to sulfate adenylyltransferase, which catalyzes a transfer reaction analogous to that of FAD synthetase. The lower mitochondrial concentration of FAD in G178 mutants is proposed to be caused by an inefficient exchange of external FAD for internal FMN. This is supported by the absence of FAD synthetase activity in yeast mitochondria and the presence of both extramitochondrial and mitochondrial riboflavin kinase, the preceding enzyme in the biosynthetic pathway. A lesion in mitochondrial import of FAD would account for the higher concentration of mitochondrial FMN in the mutant if the transport is catalyzed by an exchange carrier. The ability of FAD1 to suppress impaired transport of FAD is explained by mislocalization of the synthetase in cells harboring multiple copies of the gene. This mechanism of suppression is supported by the presence of mitochondrial FAD synthetase activity in S. cerevisiae transformed with FAD1 on a high-copy-number plasmid but not in mitochondria of a wild-type strain.Previous biochemical analyses of respiration-defective pet mutants of Saccharomyces cerevisiae (30) led to the identification of two complementation groups lacking ␣-ketoglutarate dehydrogenase (KGDC), as a result of mutations in the KGD1 and KGD2 genes for the dehydrogenase (21) and transsuccinylase (22) components of the complex, respectively. The biochemical screens also disclosed that some complementation groups in the mutant collection are composed of strains deficient in both KGDC and pyruvate dehydrogenase (PDC). Most of the mutants displaying a pleiotropic loss of both activities have been ascertained to be blocked in either the biosynthesis or attachment of lipoic acid to the apoproteins. One complementation group (G178), however, had a distinctly different phenotype. The absence of KGDC and PDC activity in G178 mutants could not be explained by the absence of lipoic acid but, rather, was correlated with a defect in lipoamide dehydrogenase, the only enzyme common to both dehydrogenase complexes (20).The results of complementation tests made it unlikely t...

Culturable vibrios were isolated from water and plankton fractions collected during an 18-month sampling study performed along the north-central coast of the Adriatic Sea (Italy). Unculturable Vibrio vulnificus and V. parahaemolyticus were detected in plankton fractions by polymerase chain reaction amplification of DNA sequences for cytotoxin-haemolysin and thermolabile haemolysin respectively. The presence of V. parahaemolyticus, V. vulnificus and V. cholerae virulence genes and the expression of pathogenicity-associated traits were analysed in all isolates. The results showed the spreading of these properties among the environmental isolates and confirm the need of both monitoring the presence of vibrios in coastal areas and studying their pathogenicity potential in order to properly protect human health.

Nuclear respiratory-defective mutants of Saccharomyces cerevisiae have been screened for lesions in the mitochondrial oa-ketoglutarate dehydrogenase complex. Strains assigned to complementation group G70 were ascertained to be deficient in enzyme activity due to mutations in the KGDI gene coding for the a-ketoglutarate dehydrogenase component of the complex. The KGDJ gene has been cloned by transformation of a representative kgdl mutant, C225/U1, with a recombinant plasmid library of wild-type yeast nuclear DNA. Transformants containing the gene on a multicopy plasmid had three-to four-times-higher a-ketoglutarate dehydrogenase activity than did wild-type S. cerevisiae. Substitution of the chromosomal copy of KGDI with a disrupted allele (kgdl::URA3) induced a deficiency in at-ketoglutarate dehydrogenase.

To examine the characteristics of the interaction of the Fc⑀RI␥ ITAM with the SH2 domains of p72 syk , the binding of an 125 I-labeled dual phosphorylated Fc⑀RI␥ ITAM-based peptide to the p72 syk SH2 domains was monitored utilizing a novel scintillation proximity based assay. The K d for this interaction, determined from the saturation binding isotherm, was 1.4 nM. This high affinity binding was reflected in the rapid rate of association for the peptide binding to the SH2 domains. Competition studies utilizing a soluble C-terminal SH2 domain knockout and N-terminal SH2 domain knockouts revealed that both domains contribute cooperatively to the high affinity binding. Unlabeled dual phosphorylated peptide competed with the 125 I-labeled peptide for binding to the dual p72 syk SH2 domains with an IC 50 value of 4.8 nM. Monophosphorylated 24-mer Fc⑀RI␥ ITAM peptides, and phosphotyrosine also competed for binding, but with substantially higher IC 50 values. This, and other data discussed, suggest that high affinity binding requires both tyrosine residues to be phosphorylated and that the preferred binding orientation of the ITAM is such that the N-terminal phosphotyrosine occupies the C-terminal SH2 domain and the C-terminal phosphotyrosine occupies the N-terminal SH2 domain. Src homology 2 (SH2)1 domains are regions of approximately 100 -120 amino acid residues present in a variety of proteins including tyrosine kinases, tyrosine phosphatases, phospholipases, and other signal transducing proteins (1-6). These domains bind with high affinity to tyrosine containing motifs in associating proteins, such as specific cytokine and immunoglobulin receptor subunits, adapter proteins, tyrosine kinases, and other signaling molecules such as STATs, when these motifs are phosphorylated by the action of tyrosine kinases (1-5, 7, 8). This allows recruitment of SH2 domain-containing signaling molecules and phosphotyrosine-containing signaling molecules into receptor-linked signal transduction assemblies (9 -13). The tyrosine-containing motifs are composed of phosphorylated tyrosine residues followed by 3-4 amino acids (e.g. pYXX(L/I)) which carry the sequence-specific information for SH2 recognition (14 -23). These motifs can occur in isolation or in tandem, thus can bind single SH2 domains (e.g. of src related tyrosine kinases) (4, 24 -26) or dual SH2 domains (e.g. of p70 zap and p72 syk ) (27-34). Certain antigen receptor subunits, such as the subunit of the T cell receptor (TCR), the Ig␣ and Ig␤ subunits of the B cell receptor and the ␤ and ␥ subunits of the high affinity IgE receptor (Fc⑀RI), contain tyrosine motifs in tandem and these have been termed immunoglobulin receptor tyrosine activation motifs (ITAMs) (24, 27, 32, 34 -36). In hematopoietic cell signaling, tyrosine-phosphorylated ITAMs have been shown to be critical for signaling interactions via their association with the SH2 domains of tyrosine kinases. For example, p70zap and the src-related kinases p59 fyn and p56 lck , appear to play a role in TCR-mediated T cell a...