The spgl gene (septum-promoting GTPase) was cloned as a multicopy suppressor of a dominant-negative mutant of the Cdc7p kinase. It encodes a small GTPase of the Ras superfamily, spgl is an essential gene. Null or heat-sensitive alleles do not make a division septum, but growth, S-phase, and mitosis continue in the absence of cell division, producing elongated, multinucleate cells. Increased expression of Spglp induces septum formation in G2, S-phase, and pre-Start Gl-arrested cells. This requires the activity of Cdc7p kinase, but not p34 cdc2. Increased expression of Cdc7p bypasses the requirement for Spglp. Spglp and Cdc7p can be coimmunoprecipitated from cell extracts, and interact in the two-hybrid system. These data indicate that Spglp is a key element in controlling the onset of septum formation in Schizosaccharomyces pombe, and that it acts through the Cdc7p kinase.
The fission yeast gene cps1, which encodes the catalytic subunit of beta-glucan synthase, was isolated in a screen for mutants that show an increase in ploidy at the restrictive temperature. cps1 mutants display defects in both polarity and septation at the permissive temperature, and become swollen and multinucleate at the restrictive temperature. Analysis of the interaction of cps1 with other mutations suggests the existence of a septation checkpoint, which requires the activity of the protein kinase weel for function.
SummaryAdhesion class G protein-coupled receptors (aGPCR) form the second largest group of seven-transmembrane-spanning (7TM) receptors whose molecular layout and function differ from canonical 7TM receptors. Despite their essential roles in immunity, tumorigenesis, and development, the mechanisms of aGPCR activation and signal transduction have remained obscure to date. Here, we use a transgenic assay to define the protein domains required in vivo for the activity of the prototypical aGPCR LAT-1/Latrophilin in Caenorhabditis elegans. We show that the GPCR proteolytic site (GPS) motif, the molecular hallmark feature of the entire aGPCR class, is essential for LAT-1 signaling serving in two different activity modes of the receptor. Surprisingly, neither mode requires cleavage but presence of the GPS, which relays interactions with at least two different partners. Our work thus uncovers the versatile nature of aGPCR activity in molecular detail and places the GPS motif in a central position for diverse protein-protein interactions.
The C. elegans pharynx is produced from the embryonic blastomeres ABa and MS. Pharyngeal fate in the ABa lineage is specified by the combined activities of GLP-1/Notch-mediated signals and the TBX-37 and TBX-38 T-box transcription factors. Here, we show another T-box factor TBX-2 also functions in ABa-derived pharyngeal development. tbx-2 mutants arrest as L1 larvae lacking most or all ABa-derived pharyngeal muscles. In comparison, tbx-2 mutants retain ABa-derived marginal cells and pharyngeal muscles derived from MS. A tbx-2Colon, two colonsgfp translational fusion is expressed in a dynamic pattern in C. elegans embryos beginning near the 100-cell stage. Early expression is limited to a small number of cells, which likely include the ABa-derived pharyngeal precursors, while later expression is observed in body wall muscles and a subset of pharyngeal neurons. TBX-2 contains 2 consensus sumoylation sites, and it interacts in a yeast two-hybrid assay with the UBC-9 and GEI-17 components of the C. elegans SUMO-conjugating pathway. ubc-9(RNAi) has been previously shown to cause variable embryonic and larval arrest, and we find that, like tbx-2 mutants, ubc-9(RNAi) animals lack ABa-derived pharyngeal muscles. ubc-9(RNAi) also alters the subnuclear distribution of TBX-2::GFP fusion protein, suggesting that UBC-9 and TBX-2 interact in C. elegans. Together, these results indicate that TBX-2 and SUMO-conjugating enzymes are necessary for ABa-derived pharyngeal muscle, and we hypothesize that TBX-2 function requires sumoylation. Sumoylation is increasingly recognized as an important mechanism controlling activity of many nuclear factors, and these results provide the first evidence that T-box factor activity may require sumoylation.
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