Antibody-free method for protein detection on blots using enzyme fragment complementation.
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Animals↗
Biomedical subjects
Joe Horecka
Publications and source records attributed to Joe Horecka.
RAM: a conserved signaling network that regulates Ace2p transcriptional activity and polarized morphogenesis.
In Saccharomyces cerevisiae, polarized morphogenesis is critical for bud site selection, bud development, and cell separation. The latter is mediated by Ace2p transcription factor, which controls the daughter cell-specific expression of cell separation genes. Recently, a set of proteins that include Cbk1p kinase, its binding partner Mob2p, Tao3p (Pag1p), and Hym1p were shown to regulate both Ace2p activity and cellular morphogenesis. These proteins seem to form a signaling network, which we designate RAM for regulation of Ace2p activity and cellular morphogenesis. To find additional RAM components, we conducted genetic screens for bilateral mating and cell separation mutants and identified alleles of the PAK-related kinase Kic1p in addition to Cbk1p, Mob2p, Tao3p, and Hym1p. Deletion of each RAM gene resulted in a loss of Ace2p function and caused cell polarity defects that were distinct from formin or polarisome mutants. Two-hybrid and coimmunoprecipitation experiments reveal a complex network of interactions among the RAM proteins, including Cbk1p-Cbk1p, Cbk1p-Kic1p, Kic1p-Tao3p, and Kic1p-Hym1p interactions, in addition to the previously documented Cbk1p-Mob2p and Cbk1p-Tao3p interactions. We also identified a novel leucine-rich repeat-containing protein Sog2p that interacts with Hym1p and Kic1p. Cells lacking Sog2p exhibited the characteristic cell separation and cell morphology defects associated with perturbation in RAM signaling. Each RAM protein localized to cortical sites of growth during both budding and mating pheromone response. Hym1p was Kic1p- and Sog2p-dependent and Sog2p and Kic1p were interdependent for localization, indicating a close functional relationship between these proteins. Only Mob2p and Cbk1p were detectable in the daughter cell nucleus at the end of mitosis. The nuclear localization and kinase activity of the Mob2p-Cbk1p complex were dependent on all other RAM proteins, suggesting that Mob2p-Cbk1p functions late in the RAM network. Our data suggest that the functional architecture of RAM signaling is similar to the S. cerevisiae mitotic exit network and Schizosaccharomyces pombe septation initiation network and is likely conserved among eukaryotes.
Cell Cycle Proteins↗
Cdc4 is involved in the transcriptional control of OCH1, a gene encoding alpha-1,6-mannosyltransferase in Saccharomyces cerevisiae.
The Saccharomyces cerevisiae OCH1 gene encodes an alpha-1,6-mannosyltransferase that initiates the polymannose outer chain elongation of N-linked glycans. Transcription of OCH1 is regulated by two transcription factors, Swi4 and Skn7. To learn more about the signals that feed into the Swi4 regulation, we isolated a mutant, bon1-1 (bypass of Skn7), that activates OCH1-HIS3 and OCH1-lacZ reporters in a strain deleted for SKN7. bon1-1 is an allele of CDC4 (cdc4(bon)) and leads to the additional phenotypes of temperature sensitivity and abnormal cell morphology. The cdc4(bon) mutant is partially suppressed by CLB5 overexpression and accumulates Sic1 protein, indicating that OCH1 transcription is controlled by the ubiquitin-dependent degradation pathway through the Skp1-Cdc53-F box(Cdc4) protein complex. We suggest that transcriptional control of OCH1 by cdc4(bon) is through Swi4, because cdc4(bon) cannot activate the OCH1-lacZ reporter in a strain deleted for SWI4. Interestingly, cdc4(bon) and Delta swi4 show a synthetic growth defect when combined.
Alleles↗
The eukaryotic two-component histidine kinase Sln1p regulates OCH1 via the transcription factor, Skn7p.
The yeast "two-component" osmotic stress phosphorelay consists of the histidine kinase, Sln1p, the phosphorelay intermediate, Ypd1p and two response regulators, Ssk1p and Skn7p, whose activities are regulated by phosphorylation of a conserved aspartyl residue in the receiver domain. Dephospho-Ssk1p leads to activation of the hyper-osmotic response (HOG) pathway, whereas phospho-Skn7p presumably leads to activation of hypo-osmotic response genes. The multifunctional Skn7 protein is important in oxidative as well as osmotic stress; however, the Skn7p receiver domain aspartate that is the phosphoacceptor in the SLN1 pathway is dispensable for oxidative stress. Like many well-characterized bacterial response regulators, Skn7p is a transcription factor. In this report we investigate the role of Skn7p in osmotic response gene activation. Our studies reveal that the Skn7p HSF-like DNA binding domain interacts with a cis-acting element identified upstream of OCH1 that is distinct from the previously defined HSE-like Skn7p binding site. Our data support a model in which Skn7p receiver domain phosphorylation affects transcriptional activation rather than DNA binding to this class of DNA binding site.
Aspartic Acid↗