The GLC7 gene of Saccharomyces cerevisiae encodes the catalytic subunit of type 1 protein phosphatase (PP1) and is essential for cell growth. We have isolated a previously uncharacterized gene, REG2, on the basis of its ability to interact with Glc7p in the two-hybrid system. Reg2p interacts with Glc7p in vivo, and epitope-tagged derivatives of Reg2p and Glc7p coimmunoprecipitate from cell extracts. The predicted protein product of the REG2 gene is similar to Reg1p, a protein believed to direct PP1 activity in the glucose repression pathway. Mutants with a deletion of reg1 display a mild slow-growth defect, while reg2 mutants exhibit a wild-type phenotype. However, mutants with deletions of both reg1 and reg2 exhibit a severe growth defect. Overexpression of REG2 complements the slow-growth defect of a reg1 mutant but does not complement defects in glycogen accumulation or glucose repression, two traits also associated with a reg1 deletion. These results indicate that REG1 has a unique role in the glucose repression pathway but acts together with REG2 to regulate some as yet uncharacterized function important for growth. The growth defect of a reg1 reg2 double mutant is alleviated by a loss-of-function mutation in the SNF1-encoded protein kinase. The snf1 mutation also suppresses the glucose repression defects of reg1. Together, our data are consistent with a model in which Reg1p and Reg2p control the activity of PP1 toward substrates that are phosphorylated by the Snf1p kinase.The reversible phosphorylation of proteins has long been recognized as a widespread mechanism of posttranslational regulation among eukaryotes. The phosphorylation state of a given protein is dependent on the relative activities of protein kinases and protein phosphatases. Early biochemical studies suggested that protein phosphatases might represent a much smaller group of enzymes than protein kinases. Whereas most kinases recognize specific motifs of five or six amino acids (46), phosphatases generally exhibit a fairly broad substrate specificity (17,64). These data contributed to the idea that cellular signaling responses were largely determined by the activities of specific protein kinases whereas phosphatases functioned at a low constitutive level (64). Recent advances have underscored the importance of protein phosphatases in controlling physiological processes and have demonstrated that protein phosphatases are in fact highly regulated.The serine/threonine protein phosphatases are among the most conserved proteins throughout evolution. The type 1 protein phosphatase (PP1) is Ͼ80% identical in mammals and in yeasts (24, 55) and has been demonstrated to play key roles in a variety of cellular processes. In mammalian cells, PP1 regulates glycogen metabolism, muscle contractility, and protein synthesis (4, 17, 64) and has been shown to interact with the product of the retinoblastoma tumor suppressor gene (20). Studies of S. cerevisiae have likewise demonstrated multiple physiological roles for PP1, including glycogen metabolism (10, 24)...

The Saccharomyces cerevisiae GLC7 gene encodes the catalytic subunit of type 1 protein phosphatase (PP1) and is required for cell growth. A cold-sensitive glc7 mutant (glc7 Y170 ) arrests in G 2 /M but remains viable at the restrictive temperature. In an effort to identify additional gene products that function in concert with PP1 to regulate growth, we isolated a mutation (gpp1) that exacerbated the growth phenotype of the glc7 Y170 mutation, resulting in rapid death of the double mutant at the nonpermissive temperature. We identified an additional gene, EGP1, as an extra-copy suppressor of the glc7 Y170 gpp1-1 double mutant. The nucleotide sequence of EGP1 predicts a leucine-rich repeat protein that is similar to Sds22, a protein from the fission yeast Schizosaccharomyces pombe that positively modulates PP1. EGP1 is essential for cell growth but becomes dispensable upon overexpression of the GLC7 gene. Egp1 and PP1 directly interact, as assayed by coimmunoprecipitation. These results suggest that Egp1 functions as a positive modulator of PP1 in the growth control of S. cerevisiae.Phosphorylation and dephosphorylation of proteins play a pivotal role in the control of diverse cellular processes, including cell cycle regulation. The phosphorylation state of a particular protein is determined by the relative activities of the protein kinases and phosphatases that recognize it as a substrate (8, 9). Since protein phosphorylation is important for the G 2 /M transition, it is expected that protein phosphatases will be shown to play an important role in this process. Indeed, studies indicate that type 1 protein phosphatase (PP1) is required for progression through mitosis. Mutations in PP1 in Schizosaccharomyces pombe (3,36), Aspergillus nidulans (14), and Drosophila melanogaster (1) cause arrest of the cell cycle in midmitosis. In mammalian cells, microinjection of an anti-PP1 antibody causes cells to arrest at metaphase (4,19). PP1 in the budding yeast Saccharomyces cerevisiae is encoded by a single essential gene, GLC7/DIS2S1 (18). Previous work suggests that in S. cerevisiae, PP1 is required for the control of mitosis as described for other organisms. We have shown that the cold-sensitive mutation glc7 Y170, which results in a cysteine-to-tyrosine substitution at position 170, causes arrest as large budded cells with undivided nuclei at the restrictive temperature (25). In mutant cells arrested at the restrictive temperature, the nucleus is positioned at or near the bud neck with a short intranuclear spindle. Furthermore, arrested cells exhibit elevated Cdc2/Cdc28 protein kinase activity. These results indicate that the glc7 Y170 mutation is defective in the G 2 /M phase of the cell cycle and that PP1 in S. cerevisiae is required for completion of mitosis.The function of PP1 in cell cycle regulation adds to its previously demonstrated roles in the regulation of glycogen and cellular metabolism, modulation of protein synthesis, and relaxation of smooth muscle (7,11,51). To explain how a single enzyme can regulate s...

Inhibitor-1 (I-1) and inhibitor-2 (I-2) selectively inhibit type 1 protein serine/threonine phosphatases (PP1). To define the molecular basis for PP1 inhibition by I-1 and I-2 charged-to-alanine substitutions in the Saccharomyces cerevisiae, PP1 catalytic subunit (GLC7), were analyzed. Two PP1 mutants, E53A/E55A and K165A/E166A/ K167A, showed reduced sensitivity to I-2 when compared with wild-type PP1. Both mutants were effectively inhibited by I-1. Two-hybrid analysis and coprecipitation or pull-down assays established that wildtype and mutant PP1 catalytic subunits bound I-2 in an identical manner and suggested a role for the mutated amino acids in enzyme inhibition. Inhibition of wildtype and mutant PP1 enzymes by full-length I-2(1-204), I-2(1-114), and I-2(36 -204) indicated that the mutant enzymes were impaired in their interaction with the Nterminal 35 amino acids of I-2. Site-directed mutagenesis of amino acids near the N terminus of I-2 and competition for PP1 binding by a synthetic peptide encompassing an I-2 N-terminal sequence suggested that a PP1 domain composed of amino acids Glu-53, Glu-55, Asp-165, Glu-166, and Lys-167 interacts with the N terminus of I-2. This defined a novel regulatory interaction between I-2 and PP1 that determines I-2 potency and perhaps selectivity as a PP1 inhibitor.

Loss-of-function gac1 mutants of Saccharomyces cerevisiae fail to accumulate normal levels of glycogen because of low glycogen synthase activity. Increased dosage of GAC1 results in increased activity of glycogen synthase and a corresponding hyperaccumulation of glycogen. The glycogen accumulation phenotype of gac1 is similar to that of glc7-1, a type 1 protein phosphatase mutant. We have partially characterized the GAC1 gene product (Gac1p) and show that levels of Gac1p increase during growth with the same kinetics as glycogen accumulation. Gac1p is phosphorylated in vivo and is hyperphosphorylated in a glc7-1 mutant. Gac1p and the type 1 protein phosphatase directly interact in vitro, as assayed by coimmunoprecipitation, and in vivo, as determined by the dihybrid assay described elsewhere (S. Fields and O.-k. Song, Nature [London] 340:245-246, 1989). The interaction between Gac1p and the glc7-1-encoded form of the type 1 protein phosphatase is defective, as assayed by either immunoprecipitation or the dihybrid assay. Increased dosage of GAC1 partially suppresses the glycogen defect of glc7-1. Collectively, our data support the hypotheses that GAC1 encodes a regulatory subunit of type 1 protein phosphatase and that the glycogen accumulation defect of glc7-1 is due at least in part to the inability of the mutant phosphatase to interact with its regulatory subunit.

Human Immunodeficiency Virus (HIV), the causative agent of Acquired Immune Deficiency Syndrome (AIDS), is a major global health concern with nearly 40 million individuals infected worldwide and no widely accessible cure. Despite intensive efforts, a detailed understanding of virus and host cell interactions in tissues during infection and in response to therapy remains incomplete. To address these limitations, water-based tissue clearing techniques CUBIC (Clear, Unobstructed Brain/Body Imaging Cocktails and Computational analysis) and CLARITY (Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging/Immunostaining/in situ-hybridization-compatible Tissue hYdrogel) are applied to visualize complex virus host-cell interactions in HIVinfected tissues from animal models and humans using confocal and light sheet fluorescence microscopy. Optical sectioning of intact tissues and image analysis allows rapid reconstruction of spatial information contained within whole tissues and quantification of immune cell populations during infection. These methods are applicable to most tissue sources and diverse biological questions, including infectious disease and cancer.