CAC3(MSI1) suppression of RAS2(G19V) is independent of chromatin assembly factor I and mediated by NPR1.
Cac3p/Msi1p, the Saccharomyces cerevisiae homolog of retinoblastoma-associated protein 48 (RbAp48), is a component of chromatin assembly factor I (CAF-I), a complex that assembles histones H3 and H4 onto replicated DNA. CAC3 overexpression also suppresses the RAS/cyclic AMP (cAMP) signal transduction pathway by an unknown mechanism. We investigated this mechanism and found that CAC3 suppression of RAS/cAMP signal transduction was independent of either CAC1 or CAC2, subunits required for CAF-I function. CAC3 suppression was also independent of other chromatin-modifying activities, indicating that Cac3p has at least two distinct, separable functions, one in chromatin assembly and one in regulating RAS function. Unlike Cac1p, which localizes primarily to the nucleus, Cac3p localizes to both the nucleus and the cytoplasm. In addition, Cac3p associates with Npr1p, a cytoplasmic kinase that stablizes several nutrient transporters by antagonizing a ubiquitin-mediated protein degradation pathway. Deletion of NPR1, like overexpression of Cac3p, suppressed the RAS/cAMP pathway. Furthermore, NPR1 overexpression interfered with the ability of CAC3 to suppress the RAS/cAMP pathway, indicating that extra Cac3p suppresses the RAS/cAMP pathway by sequestering Npr1p. Deletion of NPR1 did not affect the quantity, phosphorylation state, or localization of Ras2p. Consistent with the idea that Npr1p exerts its effect on the RAS/cAMP pathway by antagonizing a ubiquitin-mediated process, excess ubiquitin suppressed both the heat shock sensitivity and the sporulation defects caused by constitutive activation of the RAS/cAMP pathway. Thus, CAC3/MSI1 regulates the RAS/cAMP pathway via a chromatin-independent mechanism that involves the sequestration of Npr1p and may be due to the increased ubiquitination of an Npr1p substrate.
Department of Genetics, Cell Biology and Development, University of Minnesota, St. Paul, Minnesota 55108, USA.
S D
SD
Johnston
S
S
Enomoto
L
L
Schneper
M C
MC
McClellan
F
F
Twu
N D
ND
Montgomery
S A
SA
Haney
J R
JR
Broach
J
J
Berman
D000483:Alleles; D002467:Cell Nucleus / Q000378:metabolism; D056504:Chromatin Assembly Factor-1; D002868:Chromosomal Proteins, Non-Histone; D000242:Cyclic AMP / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D005690:Galactose / Q000378:metabolism; D005838:Genotype; D005947:Glucose / Q000378:metabolism; D049452:Green Fluorescent Proteins; D006358:Hot Temperature; D008164:Luminescent Proteins / Q000378:metabolism; D010641:Phenotype; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D011485:Protein Binding; D011494:Protein Kinases; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D015398:Signal Transduction; D013489:Suppression, Genetic; D013997:Time Factors; D020798:Two-Hybrid System Techniques; D018631:ras Proteins / Q000235:genetics* / Q000378:metabolism / Q000502:physiology
D056504:Chromatin Assembly Factor-1; D002868:Chromosomal Proteins, Non-Histone; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D008164:Luminescent Proteins; C061736:MSI1 protein, S cerevisiae; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D049452:Green Fluorescent Proteins; C066958:NPR1 protein, S cerevisiae; D004247:DNA; D000242:Cyclic AMP; D011494:Protein Kinases; C489015:RAS2 protein, S cerevisiae; D018631:ras Proteins; D005947:Glucose; D005690:Galactose