Purification of a <i>RAS</i>-Responsive Adenylyl Cyclase Complex from <i>Saccharomyces cerevisiae</i> by Use of an Epitope Addition Method

Field, Jeffrey; Nikawa, Jùn‐ichi; Broek, Daniel; Macdonald, Barbara S.; Rodgers, Linda; Wilson, Ian A.; Lerner, Richard A.; Wigler, Michael

doi:10.1128/mcb.8.5.2159-2165.1988

Cited by 92 publications

(36 citation statements)

References 44 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…For example, others reported copurification of Asc1 with Ras1 (in C. neoformans) and a Ras GTPase-activating protein (S. cerevisiae) [49]. Together with Gpa2 [50][51][52][53][54][55][56][57][58][59][60][61], Ras1 and Ras2 activate adenylyl cyclase [52,[62][63][64] leading to an increase in cellular cAMP [8,54,65]. Currently we are investigating novel roles of Ras1 and Ras2, using similar multi-omics approaches as those detailed above.…”

Section: Plos Geneticsmentioning

confidence: 99%

Multi-omics analysis of glucose-mediated signaling by a moonlighting Gβ protein Asc1/RACK1

Rushing

et al. 2021

PLoS Genet

View full text Add to dashboard Cite

Heterotrimeric G proteins were originally discovered through efforts to understand the effects of hormones, such as glucagon and epinephrine, on glucose metabolism. On the other hand, many cellular metabolites, including glucose, serve as ligands for G protein-coupled receptors. Here we investigate the consequences of glucose-mediated receptor signaling, and in particular the role of a Gα subunit Gpa2 and a non-canonical Gβ subunit, known as Asc1 in yeast and RACK1 in animals. Asc1/RACK1 is of particular interest because it has multiple, seemingly unrelated, functions in the cell. The existence of such “moonlighting” operations has complicated the determination of phenotype from genotype. Through a comparative analysis of individual gene deletion mutants, and by integrating transcriptomics and metabolomics measurements, we have determined the relative contributions of the Gα and Gβ protein subunits to glucose-initiated processes in yeast. We determined that Gpa2 is primarily involved in regulating carbohydrate metabolism while Asc1 is primarily involved in amino acid metabolism. Both proteins are involved in regulating purine metabolism. Of the two subunits, Gpa2 regulates a greater number of gene transcripts and was particularly important in determining the amplitude of response to glucose addition. We conclude that the two G protein subunits regulate distinct but complementary processes downstream of the glucose-sensing receptor, as well as processes that lead ultimately to changes in cell growth and metabolism.

show abstract

Section: Plos Geneticsmentioning

confidence: 99%

Multi-omics analysis of glucose-mediated signaling by a moonlighting Gβ protein Asc1/RACK1

Rushing

et al. 2021

PLoS Genet

View full text Add to dashboard Cite

show abstract

“…Adenylyl cyclase-associated protein (ACAP) was first identified in Saccharomyces cerevisiae, and CAP (adenylyl cyclase associated protein) homolog was isolated from Dictyostelium dis-coideum [12,13]. Some of the major functions of this protein involve remodeling of the actin cytoskeleton and regulating cell motility [14].…”

Section: Introductionmentioning

confidence: 99%

Characterization of a Peptide Antibody Specific to the Adenylyl Cyclase-Associated Protein of Acanthamoeba castellanii

et al. 2022

View full text Add to dashboard Cite

Acanthamoeba keratitis (AK) is a rare infectious disease and accurate diagnosis has remained arduous as clinical manifestations of AK were similar to keratitis of viral, bacterial, or fungal origins. In this study, we described the production of a polyclonal peptide antibody against the adenylyl cyclase-associated protein (ACAP) of A. castellanii, and evaluated its differential diagnostic potential. Enzyme-linked immunosorbent assay revealed high titers of A. castellanii-specific IgG and IgA antibodies being present in low dilutions of immunized rabbit serum. Western blot analysis revealed that the ACAP antibody specifically interacted with A. castellanii, while not interacting with human corneal epithelial (HCE) cells and other causes of keratitis such as Fusarium solani, Pseudomonas aeruginosa, and Staphylococcus aureus. Immunocytochemistry (ICC) results confirmed the specific detection of trophozoites and cysts of A. castellanii co-cultured with HCE cells. The ACAP antibody also specifically interacted with the trophozoites and cysts of 5 other Acanthamoeba species. These results indicate that the ACAP antibody of A. castellanii can specifically detect multiple AK-causing members belonging to the genus Acanthamoeba and may be useful for differentially diagnosing Acanthamoeba infections.

show abstract

“…This process was used to generate two mutant strains: M. mycoides subsp. capri 0582-HA, in which an HA tag ( 91 ) coding sequence is fused to the C terminus of the coding sequence of MMCAP2_0582, and M. mycoides subsp. capri mEos3.2-0575, in which the codon-optimized mEos3.2 coding sequence is fused to the N terminus of the coding sequence of MMCAP2_0575 (see the supplemental material).…”

Section: Methodsmentioning

confidence: 99%

Imaging Minimal Bacteria at the Nanoscale: a Reliable and Versatile Process to Perform Single-Molecule Localization Microscopy in Mycoplasmas

et al. 2022

View full text Add to dashboard Cite

show abstract

Purification of a RAS-Responsive Adenylyl Cyclase Complex from Saccharomyces cerevisiae by Use of an Epitope Addition Method

Cited by 92 publications

References 44 publications

Multi-omics analysis of glucose-mediated signaling by a moonlighting Gβ protein Asc1/RACK1

Multi-omics analysis of glucose-mediated signaling by a moonlighting Gβ protein Asc1/RACK1

Characterization of a Peptide Antibody Specific to the Adenylyl Cyclase-Associated Protein of Acanthamoeba castellanii

Imaging Minimal Bacteria at the Nanoscale: a Reliable and Versatile Process to Perform Single-Molecule Localization Microscopy in Mycoplasmas

Contact Info

Product

Resources

About