Interacting components of the flagellar motor of Escherichia coli revealed by the two-hybrid system in yeast.

The ability of the flagellar motor of Escherichia coli to switch between clockwise and counterclockwise modes of operation is ultimately responsible for the swimming behavior of the cell. Three motor proteins, FliG, FliM, and FliN, have been implicated in this process. Using the two-hybrid system in Saccharomyces cerevisiae, we demonstrated strong interactions between FliG/FliM,FliM/FliM, and FliM/FliN. A screen for other components that might interact with FliG revealed interactions with FliF (the MS ring protein) and H-NS (a histone-like protein). Regions of proteins important for several of these interactions were identified by mutational analysis. The implications for motor assembly and function are discussed.

A mutational analysis of the interaction between FliG and FliM, two components of the flagellar motor of Escherichia coli.

The motor that drives the flagellar filament of Escherichia coli contains three "switch" proteins (FliG, FliM, and FliN) that together determine the direction of rotation. Each is required, in addition, for flagellar assembly and for torque generation. These proteins interact in the Saccharomyces cerevisiae two-hybrid system: FliG interacts with FliM, FliM interacts with itself, and FliM interacts with FliN. The interaction between FliG and FliM has been subjected to mutational analysis. FliG (fused to the GAL4 DNA-binding domain) and FliM (fused to a GAL4 transcription activation domain) together activate transcription of a GAL4-dependent lacZ reporter gene. DNA encoding FliG was mutagenized by error-prone amplification with Taq polymerase, mutant fliG genes were cloned (as DNA-binding domain-fliG gene fusions) in S. cerevisiae by gap repair of plasmid DNA, and mutants exhibiting an interaction defect were isolated in a two-hybrid screen. The mutations were each mapped to the first, second, or last third of the fliG gene by multifragment cloning in vivo and then identified by DNA sequencing. In this way, we identified 18 interaction-defective and 15 silent (non-interaction-defective) fliG mutations. Several residues within the middle third of FliG are strongly involved in the FliG-FliM interaction, while residues near the N or C terminus are less important. This clustering, when compared with results of previous studies, suggests that the FliG-FliM interaction plays a central role in switching.

Mapping by multifragment cloning in vivo.

An efficient method for mapping mutations is described in which hybrid genes, derived partly from mutant and partly from wild-type DNA, are obtained in vivo by homologous recombination of multiple fragments. The recombinants are formed in a strain in which their phenotypes are immediately apparent. This method was developed to identify changes that disrupt protein-protein interactions demonstrable by the two-hybrid system in yeast. However, it can be extended to any system where recombination is possible, provided an assay is available to distinguish between mutant and wild-type phenotypes.

The AGA1 product is involved in cell surface attachment of the Saccharomyces cerevisiae cell adhesion glycoprotein a-agglutinin.

Saccharomyces cerevisiae a and alpha cells express the complementary cell surface glycoproteins a-agglutinin and alpha-agglutinin, respectively, which interact with one another to promote cellular aggregation during mating. Treatment of S. cerevisiae a cells with reducing agents releases the binding subunit of a-agglutinin, which has been purified and characterized; little biochemical information on the overall structure of a-agglutinin is available. To characterise a-agglutinin structure and function, we have used a genetic approach to clone an a-agglutinin structural gene (AGAI). Mutants with a-specific agglutination defects were isolated, the majority of which fell into a single complementation group, called aga1. The aga1 mutants showed wild-type pheromone production and response, efficient mating on solid medium, and a mating defect in liquid medium; these phenotypes are characteristic of agglutinin mutants. The AGA1 gene was cloned by complementation; the gene sequence indicated that it could encode a protein of 725 amino acids with high serine and threonine content, a putative N-terminal signal sequence, and a C-terminal hydrophobic sequence similar to signals for the attachment to glycosyl phosphatidylinositol anchors. Active a-agglutinin binding subunit is secreted by aga1 mutants, indicating that AGA1 is involved in cells surface attachment of a-agglutinin. This result suggests that AGA1 encodes a protein with functional similarity to the core subunits of a-agglutinin analogs from other budding yeasts. Unexpectedly, the AGA1 transcript was expressed and induced by pheromone in both a and alpha cells, suggesting that the a-specific expression of active a-agglutinin results only from a-specific regulation of the a-agglutinin binding subunit.

Control of the Saccharomyces cerevisiae regulatory gene PET494: transcriptional repression by glucose and translational induction by oxygen.

The product of the Saccharomyces cerevisiae nuclear gene PET494 is required to promote the translation of the mitochondrial mRNA encoding cytochrome c oxidase subunit III (coxIII). The level of cytochrome c oxidase activity is affected by several different environmental conditions, which also influence coxIII expression. We have studied the regulation of PET494 to test whether the level of its expression might modulate coxIII translation in response to these conditions. A pet494::lacZ fusion was constructed and used to monitor PET494 expression. PET494 was regulated by oxygen availability: expression in a respiration-competent diploid strain grown anaerobically was one-fifth the level of expression in aerobically grown cells. However, since PET494 mRNA levels did not vary in response to oxygen deprivation, regulation by oxygen appears to occur at the translational level. This oxygen regulation was not mediated by heme, and PET494 expression was independent of the heme activator protein HAP2. The regulation of PET494 expression by carbon source was also examined. In cells grown on glucose-containing medium, PET494 was expressed at levels four- to sixfold lower than in cells grown on ethanol and glycerol. However, addition of ethanol to glucose-containing medium induced PET494 expression approximately twofold. PET494 transcript levels varied over a fourfold range in response to different carbon sources. The effects on PET494 expression of mutations in the SNF1, SNF2, SSN6, and HXK2 genes were also determined and found to be minimal.

Translational regulation of mitochondrial gene expression by nuclear genes of Saccharomyces cerevisiae.

We describe several yeast nuclear mutations that specifically block expression of the mitochondrial genes encoding cytochrome c oxidase subunits II (COXII) and III (COXIII). These recessive mutations define positive regulators of mitochondrial gene expression that act at the level of translation. Mutations in the nuclear gene PET111 completely block accumulation of COXII, but the COXII mRNA is present in mutant cells at a level approximately one-third of that of the wild type. Mitochondrial suppressors of pet111 mutations correspond to deletions in mtDNA that result in fusions between the coxII structural gene and other mitochondrial genes. The chimeric mRNAs encoded by these fusions are translated in pet111 mutants; this translation leads to accumulation of functional COXII. The PET111 protein probably acts directly on coxII translation, because it is located in mitochondria. Translation of the mitochondrially coded mRNA for COXIII requires the action of at least three nuclear genes, PET494, PET54 and a newly discovered gene, provisionally termed PET55. Both the PET494 and PET54 proteins are located in mitochondria and therefore probably act directly on the mitochondrial translation system. Mutations in all three genes are suppressed in strains that contain chimeric coxIII mRNAs with the 5'-untranslated leaders of other mitochondrial transcripts fused to the coxIII coding sequence. The products of all three nuclear genes may form a complex and carry out a single function.(ABSTRACT TRUNCATED AT 250 WORDS)