scholarly journals Protein phosphatase type 1 interacts with proteins required for meiosis and other cellular processes in Saccharomyces cerevisiae.

1996 ◽  
Vol 16 (8) ◽  
pp. 4199-4206 ◽  
Author(s):  
J Tu ◽  
W Song ◽  
M Carlson
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Hybrid System ◽  
Protein Phosphatase ◽  
Regulatory Subunit ◽  
Specific Substrate ◽  
Type I ◽  
Cellular Processes ◽  
Cell Extracts ◽  
Two Hybrid ◽  
Protein Phosphatase Type

Protein phosphatase type I (PP1) is involved in diverse cellular processes, and its activity toward specific substrates is thought to be controlled by different regulatory or targeting subunits. To identify regulatory subunits and substrates of the Saccharomyces cerevisiae PP1, encoded by GLC7, we used the two-hybrid system to detect interacting proteins. Among the many proteins identified were Gac1, a known glycogen regulatory subunit, and a protein with homology to Gac1. We also characterized a new gene designated GIP1, for Glc7-interacting protein. We show that a Gip1 fusion protein coimmunoprecipitates with PP1 from cell extracts. Molecular and genetic analyses indicate that GIP1 is expressed specifically during meiosis, affects transcription of late meiotic genes, and is essential for sporulation. Thus, the Gip1 protein is a candidate for a meiosis-specific substrate or regulator of PP1. Finally, we recovered two genes, RED1 and SCD5, with roles in meiosis and the vesicular secretory pathway, respectively. These results provide strong evidence implicating PP1 function in meiosis. In addition, this study indicates that the two-hybrid system offers a promising approach to understanding the multiple roles and interactions of PP1 in cellular regulation.

scholarly journals Sip5 Interacts With Both the Reg1/Glc7 Protein Phosphatase and the Snf1 Protein Kinase of Saccharomyces cerevisiae

Genetics ◽  
2000 ◽  
Vol 154 (1) ◽  
pp. 99-107
Author(s):  
Pascual Sanz ◽  
Katja Ludin ◽  
Marian Carlson
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Kinase ◽  
Protein Phosphatase ◽  
Regulatory Subunit ◽  
Glucose Starvation ◽  
Glucose Limitation ◽  
Snf1 Kinase ◽  
Cell Extracts ◽  
Two Hybrid ◽  
New Protein

Abstract The Snf1 protein kinase is an essential component of the glucose starvation signalling pathway in Saccharomyces cerevisiae. We have used the two-hybrid system to identify a new protein, Sip5, that interacts with the Snf1 kinase complex in response to glucose limitation. Coimmunoprecipitation studies confirmed the association of Sip5 and Snf1 in cell extracts. We found that Sip5 also interacts strongly with Reg1, the regulatory subunit of the Reg1/Glc7 protein phosphatase 1 complex, in both two-hybrid and coimmunoprecipitation assays. Previous work showed that Reg1/Glc7 interacts with the Snf1 kinase under glucose-limiting conditions and negatively regulates its activity. Sip5 is the first protein that has been shown to interact with both Snf1 and Reg1/Glc7. Genetic analysis showed that the two-hybrid interaction between Reg1 and Snf1 is reduced threefold in a sip5Δ mutant. These findings suggest that Sip5 facilitates the interaction between the Reg1/Glc7 phosphatase and the Snf1 kinase.

Interaction of the Repressors Nrg1 and Nrg2 With the Snf1 Protein Kinase in Saccharomyces cerevisiae

Genetics ◽  
2001 ◽  
Vol 158 (2) ◽  
pp. 563-572 ◽  
Author(s):  
Valmik K Vyas ◽  
Sergei Kuchin ◽  
Marian Carlson
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Kinase ◽  
Hybrid System ◽  
Fluorescent Protein ◽  
Zinc Finger Protein ◽  
Glucose Repression ◽  
Snf1 Kinase ◽  
Cell Extracts ◽  
Green Fluorescent ◽  
Two Hybrid

Abstract The Snf1 protein kinase is essential for the transcription of glucose-repressed genes in Saccharomyces cerevisiae. We identified Nrg2 as a protein that interacts with Snf1 in the two-hybrid system. Nrg2 is a C2H2 zinc-finger protein that is homologous to Nrg1, a repressor of the glucose- and Snf1-regulated STA1 (glucoamylase) gene. Snf1 also interacts with Nrg1 in the two-hybrid system and co-immunoprecipitates with both Nrg1 and Nrg2 from cell extracts. A LexA fusion to Nrg2 represses transcription from a promoter containing LexA binding sites, indicating that Nrg2 also functions as a repressor. An Nrg1 fusion to green fluorescent protein is localized to the nucleus, and this localization is not regulated by carbon source. Finally, we show that VP16 fusions to Nrg1 and Nrg2 allow low-level expression of SUC2 in glucose-grown cells, and we present evidence that Nrg1 and Nrg2 contribute to glucose repression of the DOG2 gene. These results suggest that Nrg1 and Nrg2 are direct or indirect targets of the Snf1 kinase and function in glucose repression of a subset of Snf1-regulated genes.

scholarly journals Dynamic Localization of Protein Phosphatase Type 1 in the Mitotic Cell Cycle of Saccharomyces cerevisiae

2000 ◽  
Vol 149 (1) ◽  
pp. 125-140 ◽  
Author(s):  
Andrew Bloecher ◽  
Kelly Tatchell
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Protein Phosphatase ◽  
Essential Gene ◽  
Mitotic Cell ◽  
Type I ◽  
Mitotic Cell Cycle ◽  
Dependent Manner ◽  
Bud Neck ◽  
Protein Phosphatase Type

Protein phosphatase type I (PP1), encoded by the single essential gene GLC7 in Saccharomyces cerevisiae, functions in diverse cellular processes. To identify in vivo subcellular location(s) where these processes take place, we used a functional green fluorescent protein (GFP)–Glc7p fusion protein. Time-lapse fluorescence microscopy revealed GFP–Glc7p localizes predominantly in the nucleus throughout the mitotic cell cycle, with the highest concentrations in the nucleolus. GFP–Glc7p was also observed in a ring at the bud neck, which was dependent upon functional septins. Supporting a role for Glc7p in bud site selection, a glc7-129 mutant displayed a random budding pattern. In α-factor treated cells, GFP–Glc7p was located at the base of mating projections, again in a septin-dependent manner. At the start of anaphase, GFP–Glc7p accumulated at the spindle pole bodies and remained there until cytokinesis. After anaphase, GFP–Glc7p became concentrated in a ring that colocalized with the actomyosin ring. A GFP–Glc7-129 fusion was defective in localizing to the bud neck and SPBs. Together, these results identify sites of Glc7p function and suggest Glc7p activity is regulated through dynamic changes in its location.

scholarly journals Interaction of a Swi3 homolog with Sth1 provides evidence for a Swi/Snf-related complex with an essential function in Saccharomyces cerevisiae.

1997 ◽  
Vol 17 (4) ◽  
pp. 1768-1775 ◽  
Author(s):  
I Treich ◽  
M Carlson
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Hybrid System ◽  
Transcriptional Activation ◽  
Leucine Zipper ◽  
Leucine Zipper Motif ◽  
Cell Extracts ◽  
Essential Function ◽  
Self Association ◽  
Two Hybrid

The Saccharomyces cerevisiae Swi/Snf complex has a role in remodeling chromatin structure to facilitate transcriptional activation. The complex has 11 components, including Swi1/Adr6, Swi2/Snf2, Swi3, Snf5, Snf6, Snf11, Swp73/Snf12, and Tfg3. Mammalian homologs of these proteins have been shown to form multiple Swi/Snf-related complexes. Here we characterize an S. cerevisiae Swi3 homolog (Swh3) and present evidence that it associates in a complex with a Snf2 homolog, Sthl. We identified Swh3 as a protein that interacts with the N terminus of Snf2 in the two-hybrid system. Swh3 and Swi3 are functionally distinct, and overexpression of one does not compensate for loss of the other. Swh3 is essential for viability and does not activate transcription of reporters. The Snf2 sequence that interacts with Swh3 was mapped to a region conserved in Sth1. We show that Swh3 and Sth1 fusion proteins interact in the two-hybrid system and coimmunoprecipitate from yeast cell extracts. We also map interactions between Swh3 and Sth1 and examine the role of a leucine zipper motif in self-association of Swh3. These findings, together with previous analysis of Sth1, indicate that Swh3 and Sth1 are associated in a complex that is functionally distinct from the Swi/Snf complex and essential for viability.

scholarly journals GAC1 may encode a regulatory subunit for protein phosphatase type 1 in Saccharomyces cerevisiae.

1992 ◽  
Vol 11 (1) ◽  
pp. 87-96 ◽  
Author(s):  
J.M. François ◽  
S. Thompson-Jaeger ◽  
J. Skroch ◽  
U. Zellenka ◽  
W. Spevak ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Phosphatase ◽  
Regulatory Subunit ◽  
Protein Phosphatase Type 1 ◽  
Protein Phosphatase Type

scholarly journals Ectopic Expression of Inhibitors of Protein Phosphatase Type 1 (PP1) Can Be Used to Analyze Roles of PP1 in Drosophila Development

Genetics ◽  
2003 ◽  
Vol 164 (1) ◽  
pp. 235-245
Author(s):  
Daimark Bennett ◽  
Balázs Szöőr ◽  
Sascha Gross ◽  
Natalia Vereshchagina ◽  
Luke Alphey
Keyword(s):  
Protein Phosphatase ◽  
Muscle Development ◽  
Ectopic Expression ◽  
High Specificity ◽  
Type I ◽  
Protein Phosphatase Type 1 ◽  
Mitotic Figures ◽  
Protein Phosphatase Type

Abstract We have identified two proteins that bind with high specificity to type 1 serine/threonine protein phosphatase (PP1) and have exploited their inhibitory properties to develop an efficient and flexible strategy for conditional inactivation of PP1 in vivo. We show that modest overexpression of Drosophila homologs of I-2 and NIPP1 (I-2Dm and NIPP1Dm) reduces the level of PP1 activity and phenotypically resembles known PP1 mutants. These phenotypes, which include lethality, abnormal mitotic figures, and defects in muscle development, are suppressed by coexpression of PP1, indicating that the effect is due specifically to loss of PP1 activity. Reactivation of I-2Dm:PP1c complexes suggests that inhibition of PP1 activity in vivo does not result in a compensating increase in synthesis of active PP1. PP1 mutants enhance the wing overgrowth phenotype caused by ectopic expression of the type II TGFβ superfamily signaling receptor Punt. Using I-2Dm, which has a less severe effect than NIPP1Dm, we show that lowering the level of PP1 activity specifically in cells overexpressing Punt is sufficient for wing overgrowth and that the interaction between PP1 and Punt requires the type I receptor Thick-veins (Tkv) but is not strongly sensitive to the level of the ligand, Decapentaplegic (Dpp), nor to that of the other type I receptors. This is consistent with a role for PP1 in antagonizing Punt by preventing phosphorylation of Tkv. These studies demonstrate that inhibitors of PP1 can be used in a tissue- and developmental-specific manner to examine the developmental roles of PP1.

scholarly journals The split protein phosphatase system

2018 ◽  
Vol 475 (23) ◽  
pp. 3707-3723 ◽  
Author(s):  
Anne Bertolotti
Keyword(s):  
Protein Phosphatase ◽  
Catalytic Subunit ◽  
Regulatory Subunit ◽  
Post Translational Modification ◽  
Antagonistic Action ◽  
Cellular Processes ◽  
Depth Study ◽  
Split Protein ◽  
Phosphatase System ◽  
Physiological Substrates

Reversible phosphorylation of proteins is a post-translational modification that regulates all aspect of life through the antagonistic action of kinases and phosphatases. Protein kinases are well characterized, but protein phosphatases have been relatively neglected. Protein phosphatase 1 (PP1) catalyzes the dephosphorylation of a major fraction of phospho-serines and phospho-threonines in cells and thereby controls a broad range of cellular processes. In this review, I will discuss how phosphatases were discovered, how the view that they were unselective emerged and how recent findings have revealed their exquisite selectivity. Unlike kinases, PP1 phosphatases are obligatory heteromers composed of a catalytic subunit bound to one (or two) non-catalytic subunit(s). Based on an in-depth study of two holophosphatases, I propose the following: selective dephosphorylation depends on the assembly of two components, the catalytic subunit and the non-catalytic subunit, which serves as a high-affinity substrate receptor. Because functional complementation of the two modules is required to produce a selective holophosphatase, one can consider that they are split enzymes. The non-catalytic subunit was often referred to as a regulatory subunit, but it is, in fact, an essential component of the holoenzyme. In this model, a phosphatase and its array of mostly orphan substrate receptors constitute the split protein phosphatase system. The set of potentially generalizable principles outlined in this review may facilitate the study of these poorly understood enzymes and the identification of their physiological substrates.

scholarly journals Mutations in GCR1, a transcriptional activator of Saccharomyces cerevisiae glycolytic genes, function as suppressors of gcr2 mutations.

Genetics ◽  
1995 ◽  
Vol 139 (2) ◽  
pp. 511-521 ◽  
Author(s):  
H Uemura ◽  
Y Jigami
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Hybrid System ◽  
Enzyme Assay ◽  
Transcriptional Activator ◽  
Activation Domain ◽  
Specific Mutation ◽  
Activating Function ◽  
Affect Expression ◽  
Two Hybrid

Abstract The Saccharomyces cerevisiae GCR1 and GCR2 genes affect expression of most of the glycolytic genes. Evidence for Gcr1p/Gcr2p interaction has been presented earlier and is now supported by the isolation of mutations in Gcr1p suppressing gcr2, as assessed by growth and enzyme assay. Four specific mutation sites were identified. Together with use of the two-hybrid system of Fields and Song, they show that Gcr1p in its N-terminal half has a potential transcriptional activating function as well as elements for interaction with Gcr2p, which perhaps acts normally to expose an otherwise cryptic activation domain on Gcr1p. Complementation of various gcr1 mutant alleles and results with the two-hybrid system also indicate that Gcr1p itself normally functions as an oligomer.

Sign in / Sign up

Export Citation Format

Share Document