Glycogen-binding Domain Research Articles

The Aspergillus nidulans esdC (early sexual development) gene is necessary for sexual development and is controlled by veA and a heterotrimeric G protein

The esdC ( early sexual development) gene was isolated by using an expressed sequence tag (EST) as a probe from a genomic library of the early sexual developmental stage mycelia of Aspergillus nidulans. The sequence analysis revealed that the esdC gene contains a 59 bp intron and encodes a 266 amino acid polypeptide with a calculated molecular weight of 29.4 kDa. The EsdC protein is conserved among filamentous fungi and has a domain with similarity to a glycogen binding domain conserved in the β subunit of the AMP-activated protein kinase (AMPK) complex. Although the esdD gene was expressed during asexual development, the expression reached its maximum at 10 h and decreased thereafter up to 50 h after the end of the induction of sexual development. In an esdC-null mutant under a veA + background, no sexual structures were formed at any condition examined. However, esdC overexpression did not lead to an induction of sexual development. In addition, to the effect of the esdC mutation on the sexual development, more conidiophores were formed in the esdC-null mutant than in a wild type. These results indicate that the esdC gene is necessary for sexual structure formation but its overexpression is not sufficient to enhance this process. Expression of the esdC gene throughout development was positively regulated by the veA gene. In addition, very little and no esdC transcript, respectively, was observed in an flbA-null mutant and in a fadA G42R mutant, and the esdC transcript level was higher in a fadA-null mutant and in a sfaD-null mutant than in a wild type, indicating that inactivation of FadA is necessary for positive regulation of esdC expression.

Crystal structure of the heterotrimer core of Saccharomyces cerevisiae AMPK homologue SNF1

AMP-activated protein kinase (AMPK) is a central regulator of energy homeostasis in mammals and is an attractive target for drug discovery against diabetes, obesity and other diseases. The AMPK homologue in Saccharomyces cerevisiae, known as SNF1, is essential for responses to glucose starvation as well as for other cellular processes, although SNF1 seems to be activated by a ligand other than AMP. Here we report the crystal structure at 2.6 A resolution of the heterotrimer core of SNF1. The ligand-binding site in the gamma-subunit (Snf4) has clear structural differences from that of the Schizosaccharomyces pombe enzyme, although our crystallographic data indicate that AMP can also bind to Snf4. The glycogen-binding domain in the beta-subunit (Sip2) interacts with Snf4 in the heterotrimer but should still be able to bind carbohydrates. Our structure is supported by a large body of biochemical and genetic data on this complex. Most significantly, the structure reveals that part of the regulatory sequence in the alpha-subunit (Snf1) is sequestered by Snf4, demonstrating a direct interaction between the alpha- and gamma-subunits and indicating that our structure may represent the heterotrimer core of SNF1 in its activated state.

AMP-activated protein kinase does not associate with glycogen α-particles from rat liver

The AMP-activated protein kinase (AMPK) is heterotrimer consisting of α catalytic subunit and β/γ regulatory subunits. It acts as a critical focal point for whole body and cellular mechanisms maintaining energy homeostasis by regulating carbohydrate and lipid metabolism, food intake, gene transcription, and protein synthesis. The AMPK β subunit contains a glycogen-binding domain that has been shown to associate with glycogen particles in vitro and glycogen phosphorylase and glycogen synthase in cultured cells. To determine whether AMPK associates with glycogen particles in vivo, we developed a procedure to purify glycogen α-particles to apparent homogeneity from rat liver. Using immunoreactivity and mass spectrometry we determined that AMPK does not associate with the glycogen particle in livers from random-fed rats. This surprising finding indicates that the glycogen-binding properties of the AMPK β subunit are likely regulated and responsive to the metabolic status of the hepatocyte.

Association of AMP kinase β 2 subunit with glycogen particles as revealed by immunoelectron microscopy

AMP kinase β2 subunit contains a functional glycogen binding domain (β-GBD) that links AMPK to glycogen metabolism. By routine electron microscopy, glycogen deposits in cells are easily visualized as small rosettes of dark granules. However, immunocytochemistry requires weak fixation protocols that allow preservation of protein antigenicity but have the drawback of preventing retention of certain cellular elements such as glycogen. The cells thus appear vacuolated. Immunogold for AMPK subunits on such tissues leads to specific labelings located only in cytoplasmic areas surrounding the endoplasmic reticulum. We herein report the development of a new protocol for immunoelectron microscopy that retains glycogen deposits within the cells with the possibility of performing successfully the immunolabeling for the AMPK subunits. Upon applying specific antibodies against the various AMPK subunits on rat liver tissue, quantitative immunogold revealed that the labeling for the β2 subunit is closely associated, for its very large majority, with the glycogen particles of the cell. The other subunits, α1 and α2, are more abundant in cytoplasmic areas close to the endoplasmic reticulum and mitochondria. These results demonstrate the existence of a structural link between AMPK β2 subunit and glycogen, a major energy store of the cell.

AKINβγ Contributes to SnRK1 Heterotrimeric Complexes and Interacts with Two Proteins Implicated in Plant Pathogen Resistance through Its KIS/GBD Sequence

The sucrose nonfermenting-1 protein kinase (SNF1)/AMP-activated protein kinase subfamily plays a central role in metabolic responses to nutritional and environmental stresses. In yeast (Saccharomyces cerevisiae) and mammals, the beta- and gamma-noncatalytic subunits are implicated in substrate specificity and subcellular localization, respectively, and regulation of the kinase activity. The atypical betagamma-subunit has been previously described in maize (Zea mays), presenting at its N-terminal end a sequence related to the KIS (kinase interacting sequence) domain specific to the beta-subunits (Lumbreras et al., 2001). The existence of two components, SNF1-related protein kinase (SnRK1) complexes containing the betagamma-subunit and one SnRK1 kinase, had been proposed. In this work, we show that, despite its unusual features, the Arabidopsis (Arabidopsis thaliana) homolog AKINbetagamma clearly interacts with AKINbeta-subunits in vitro and in vivo, suggesting its involvement in heterotrimeric complexes located in both cytoplasm and nucleus. Unexpectedly, a transcriptional analysis of AKINbetagamma gene expression highlighted the implication of alternative splicing mechanisms in the regulation of AKINbetagamma expression. A two-hybrid screen performed with AKINbetagamma as bait, together with in planta bimolecular fluorescence complementation experiments, suggests the existence of interactions in the cytosol between AKINbetagamma and two leucine-rich repeats related to pathogen resistance proteins. Interestingly, this interaction occurs through the truncated KIS domain that corresponds exactly to a GBD (glycogen-binding domain) recently described in mammals and yeast. A phylogenetic study suggests that AKINbetagamma-related proteins are restricted to the plant kingdom. Altogether, these data suggest the existence of plant-specific SnRK1 trimeric complexes putatively involved in a plant-specific function such as plant-pathogen interactions.

Relationship between glycogen accumulation and the laforin dual specificity phosphatase

Laforin, encoded by the EPM2A gene, is a dual specificity protein phosphatase that has a functional glycogen-binding domain. Mutations in the EPM2A gene account for around half of the cases of Lafora disease, an autosomal recessive neurodegenerative disorder, characterized by progressive myoclonus epilepsy. The hallmark of the disease is the presence of Lafora bodies, which contain polyglucosan, a poorly branched form of glycogen, in neurons and other tissues. We examined the level of laforin protein in several mouse models in which muscle glycogen accumulation has been altered genetically. Mice with elevated muscle glycogen have increased laforin as judged by Western analysis. Mice completely lacking muscle glycogen or with 10% normal muscle glycogen had reduced laforin. Mice defective in the GAA gene encoding lysosomal α-glucosidase (acid maltase) overaccumulate glycogen in the lysosome but did not have elevated laforin. We propose, therefore, that laforin senses cytosolic glycogen accumulation which in turn determines the level of laforin protein.

Reduced glycogen availability is associated with increased AMPKα2 activity, nuclear AMPKα2 protein abundance, and GLUT4 mRNA expression in contracting human skeletal muscle

Glycogen availability can influence glucose transporter 4 (GLUT4) expression in skeletal muscle through unknown mechanisms. The multisubstrate enzyme AMP-activated protein kinase (AMPK) has also been shown to play an important role in the regulation of GLUT4 expression in skeletal muscle. During contraction, AMPK alpha2 translocates to the nucleus and the activity of this AMPK isoform is enhanced when skeletal muscle glycogen is low. In this study, we investigated if decreased pre-exercise muscle glycogen levels and increased AMPK alpha2 activity reduced the association of AMPK with glycogen and increased AMPK alpha2 translocation to the nucleus and GLUT4 mRNA expression following exercise. Seven males performed 60 min of exercise at approximately 70% VO(2) (peak) on 2 occasions: either with normal (control) or low (LG) carbohydrate pre-exercise muscle glycogen content. Muscle samples were obtained by needle biopsy before and after exercise. Low muscle glycogen was associated with elevated AMPK alpha2 activity and acetyl-CoA carboxylase beta phosphorylation, increased translocation of AMPK alpha2 to the nucleus, and increased GLUT4 mRNA. Transfection of primary human myotubes with a constitutively active AMPK adenovirus also stimulated GLUT4 mRNA, providing direct evidence of a role of AMPK in regulating GLUT4 expression. We suggest that increased activation of AMPK alpha2 under conditions of low muscle glycogen enhances AMPK alpha2 nuclear translocation and increases GLUT4 mRNA expression in response to exercise in human skeletal muscle.

A chloroplast‐localized dual‐specificity protein phosphatase in Arabidopsis contains a phylogenetically dispersed and ancient carbohydrate‐binding domain, which binds the polysaccharide starch

Dual-specificity protein phosphatases (DSPs) are important regulators of a wide variety of protein kinase signaling cascades in animals, fungi and plants. We previously identified a cluster of putative DSPs in Arabidopsis (including At3g52180 and At3g01510) in which the phosphatase domain is related to that of laforin, the human protein mutated in Lafora epilepsy. In animal and fungal systems, the laforin DSP and the beta-regulatory subunits of AMP-regulated protein kinase (AMPK) and Snf-1 have all been demonstrated to bind to glycogen by a glycogen-binding domain (GBD). We present a bioinformatic analysis which shows that these DSPs from Arabidopsis, together with other related plant DSPs, share with the above animal and fungal proteins a widespread and ancient carbohydrate-binding domain. We demonstrate that DSP At3g52180 binds to purified starch through its predicted carbohydrate-binding region, and that mutation of key conserved residues reduces this binding. Consistent with its ability to bind exogenous starch, DSP At3g52180 was found associated with starch purified from Arabidopsis plants and suspension cells. Immunolocalization experiments revealed a co-localization with chlorophyll, placing DSP At3g52180 in the chloroplast. Gene-expression data from different stages of the light-dark cycle and across a wide variety of tissues show a strong correlation between the pattern displayed by transcripts of the At3g52180 locus and that of genes encoding key starch degradative enzymes. Taken together, these data suggest the hypothesis that plant DSPs could be part of a protein assemblage at the starch granule, where they would be ideally situated to regulate starch metabolism through reversible phosphorylation events.

Structural Basis for Glycogen Recognition by AMP-Activated Protein Kinase

AMP-activated protein kinase (AMPK) coordinates cellular metabolism in response to energy demand as well as to a variety of stimuli. The AMPK beta subunit acts as a scaffold for the alpha catalytic and gamma regulatory subunits and targets the AMPK heterotrimer to glycogen. We have determined the structure of the AMPK beta glycogen binding domain in complex with beta-cyclodextrin. The structure reveals a carbohydrate binding pocket that consolidates all known aspects of carbohydrate binding observed in starch binding domains into one site, with extensive contact between several residues and five glucose units. beta-cyclodextrin is held in a pincer-like grasp with two tryptophan residues cradling two beta-cyclodextrin glucose units and a leucine residue piercing the beta-cyclodextrin ring. Mutation of key beta-cyclodextrin binding residues either partially or completely prevents the glycogen binding domain from binding glycogen. Modeling suggests that this binding pocket enables AMPK to interact with glycogen anywhere across the carbohydrate's helical surface.

AMP-activated Protein Kinase β Subunit Tethers α and γ Subunits via Its C-terminal Sequence (186–270)

AMP-activated protein kinase (AMPK) is an important metabolic stress-sensing protein kinase responsible for regulating metabolism in response to changing energy demand and nutrient supply. Mammalian AMPK is a stable αβγ heterotrimer comprising a catalytic α and two non-catalytic subunits, β and γ. The β subunit targets AMPK to membranes via an N-terminal myristoyl group and to glycogen via a mid-molecule glycogen-binding domain. Here we find that the conserved C-terminal 85-residue sequence of the β subunit, β1-(186–270), is sufficient to form an active AMP-dependent heterotrimer α1β1-(186–270)-γ1, whereas the 25-residue β1 C-terminal (246–270) sequence is sufficient to bind γ1, γ2, or γ3 but not the α subunit. Deletion of the β C-terminal Ile-270 precludes βγ association in the absence of the α subunit, but the presence of the α subunit or substitution of Ile-270 with Ala or Glu restores βγ binding. Truncation of the α subunit reveals that β1 binding requires the α1-(313–473) sequence. The conserved C-terminal 85-residue sequence of the β subunit (90% between β1 and β2) is the primary αγ binding sequence responsible for the formation of the AMPK αβγ heterotrimer.

AKIN?3, a plant specific SnRK1 protein, is lacking domains present in yeast and mammals non-catalytic ?-subunits

The SNF1/AMPK/SnRK1 heterotrimeric kinase complex is involved in the adaptation of cellular metabolism in response to diverse stresses in yeast, mammals and plants. Following a model proposed in yeast, the kinase targets are likely to bind the complex via the non-catalytic beta-subunits. These proteins currently identified in yeast, mammals and plants present a common structure with two conserved interacting domains named Kinase Interacting Sequence (KIS) and Association with SNF1 Complex (ASC), and a highly variable N-terminal domain. In this paper we describe the characterisation of AKINbeta3, a novel protein related to AKINbeta subunits of Arabidopsis thaliana, containing a truncated KIS domain and no N-terminal extension. Interestingly the missing region of the KIS domain corresponds to the glycogen-binding domain (beta-GBD) identified in the mammalian AMPKbeta1. In spite of its unusual features, AKINbeta3 complements the yeast sip1Deltasip2Deltagal83Delta mutant. Moreover, interactions between AKINbeta3 and other AKIN complex subunits from A. thaliana were detected by two-hybrid experiments and in vitro binding assays. Taken together these data demonstrate that AKINbeta3 is a beta-type subunit. A search for beta-type subunits revealed the existence of beta3-type proteins in other plant species. Furthermore, we suggest that the AKINbeta3-type subunits could be plant specific since no related sequences have been found in any of the other completely sequenced genomes. These data suggest the existence of novel SnRK1 complexes including AKINbeta3-type subunits, involved in several functions among which some could be plant specific.

Crystallization of the glycogen-binding domain of the AMP-activated protein kinase beta subunit and preliminary X-ray analysis.

AMP-activated protein kinase (AMPK) is an intracellular energy sensor that regulates metabolism in response to energy demand and supply by adjusting the ATP-generating and ATP-consuming pathways. AMPK potentially plays a critical role in diabetes and obesity as it is known to be activated by metforin and rosiglitazone, drugs used for the treatment of type II diabetes. AMPK is a heterotrimer composed of a catalytic alpha subunit and two regulatory subunits, beta and gamma. Mutations in the gamma subunit are known to cause glycogen accumulation, leading to cardiac arrhythmias. Recently, a functional glycogen-binding domain (GBD) has been identified in the beta subunit. Here, the crystallization of GBD in the presence of beta-cyclodextrin is reported together with preliminary X-ray data analysis allowing the determination of the structure by single isomorphous replacement and threefold averaging using in-house X-ray data collected from a selenomethionine-substituted protein.

AMPK

First discovered… AMP-activated protein kinase (AMPK) was first named in 1988 although it had appeared in several earlier reports under a number of different aliases, e.g. HMG-CoA reductase kinase, acetyl-CoA carboxylase kinase. The name AMP-activated protein kinase was chosen after one of its physiological activators, AMP. Don't ever say… Cyclic-AMP dependent protein kinase in the same breath! AMPK and PKA may have similar-sounding names but they are regulated by different pathways and their downstream effects, although in some cases overlap, are quite distinct. What does it do? AMPK has been dubbed the cellular fuel gauge, because it is activated by a drop in the energy status of the cell. If ATP is used up faster than it can be re-synthesized, ATP levels fall and this leads to a rise in AMP. The increase in the AMP:ATP ratio triggers the activation of AMPK and leads to the phosphorylation of a large number of downstream targets. The overall effect of AMPK activation is to switch off energy-using pathways and switch on energy-generating pathways, thus helping to restore the energy balance within the cell. The conservation of AMPK throughout evolution emphasises its importance: homologs have been identified in all eukaryotic species examined to date, including plants. More recent findings have shown that AMPK is activated by stimuli that do not cause a change in the AMP:ATP ratio, such as hyperosmotic stress, indicating that the kinase may have other roles within the cell. What does it look like? AMPK is a heterotrimeric enzyme complex. The α subunit contains a typical serine/threonine kinase domain and a carboxy-terminal regulatory domain. The β subunit acts as a scaffold for binding the other two subunits and contains a glycogen-binding domain. The γ subunit contains four cystathionine-β-synthase (CBS) domains that play a role in binding AMP and ATP, allosteric effectors of the kinase. A range of isoforms of each of the subunits results in 12 possible combinations of the heterotrimeric complex in mammals. The physiological role of the different isoforms is not clear, although their differing tissue distributions suggest they have distinct roles in specific cells. How is it regulated? Activation of AMPK requires phosphorylation by an upstream kinase, which was recently identified as LKB1. Intriguingly, inactivating mutations in LKB1 cause a rare form of hereditary cancer, called Peutz-Jeghers syndrome. It is not yet clear whether AMPK is involved in the progression of this disease, but it does hint at the possibility that AMPK may be involved in cell proliferation. Can we live without it? Almost certainly not. Mice lacking either one of the catalytic α subunits are viable, but deleting both isoforms results in embryonic lethality. Interestingly, mice lacking LKB1 also die at around the same stage during development. Although a complete loss of AMPK activity is lethal, subtle changes in AMPK activity can lead to serious clinical consequences. Naturally occurring mutations within the γ2 regulatory subunit in humans cause cardiac hypertrophy associated with a form of arrhythmia known as Wolff–Parkinson–White syndrome, possibly by abolishing the normal activation of the kinase by AMP. Where can I find out more?

Mutations in the gal83 glycogen-binding domain activate the snf1/gal83 kinase pathway by a glycogen-independent mechanism.

The yeast Snf1 kinase and its mammalian ortholog, AMP-activated protein kinase (AMPK), regulate responses to metabolic stress. Previous studies identified a glycogen-binding domain in the AMPK beta1 subunit, and the sequence is conserved in the Snf1 kinase beta subunits Gal83 and Sip2. Here we use genetic analysis to assess the role of this domain in vivo. Alteration of Gal83 at residues that are important for glycogen binding of AMPK beta1 abolished glycogen binding in vitro and caused diverse phenotypes in vivo. Various Snf1/Gal83-dependent processes were upregulated, including glycogen accumulation, expression of RNAs encoding glycogen synthase, haploid invasive growth, the transcriptional activator function of Sip4, and activation of the carbon source-responsive promoter element. Moreover, the glycogen-binding domain mutations conferred transcriptional regulatory phenotypes even in the absence of glycogen, as determined by analysis of a mutant strain lacking glycogen synthase. Thus, mutation of the glycogen-binding domain of Gal83 positively affects Snf1/Gal83 kinase function by a mechanism that is independent of glycogen binding.

Management of cellular energy by the AMP-activated protein kinase system

The AMP-activated protein kinase is a sensor of cellular energy status that is found in all eukaryotic cells. It is activated by rising AMP and falling ATP by a complex mechanism that results in an ultrasensitive response. The functions of the different domains on the three subunits of the αβγ heterotrimer are slowly being unravelled, and a recent development has been the identification of a glycogen-binding domain on the β subunit. Along with findings that high cellular glycogen represses kinase activation, this suggests that the system may be a sensor of glycogen content as well as of AMP and ATP. New insights have been obtained into the sequence and structural features by which the kinase recognises its downstream target proteins, and these are discussed. Once activated by depletion of cellular energy reserves, the kinase switches on ATP-producing catabolic pathways and switches off ATP-consuming processes, both via direct phosphorylation of regulatory proteins and via indirect effects on gene expression. A survey of the range of downstream targets for this important signalling pathway is presented.

A Novel Domain in AMP-Activated Protein Kinase Causes Glycogen Storage Bodies Similar to Those Seen in Hereditary Cardiac Arrhythmias

The AMP-activated protein kinase (AMPK) is an αβγ heterotrimer that is activated by low cellular energy status and affects a switch away from energy-requiring processes and toward catabolism [1]. While it is primarily regulated by AMP and ATP, high muscle glycogen has also been shown to repress its activation [2, 3]. Mutations in the γ2 and γ3 subunit isoforms lead to arrhythmias associated with abnormal glycogen storage in human heart [4–7] and elevated glycogen in pig muscle [8], respectively. A putative glycogen binding domain (GBD) has now been identified in the β subunits. Coexpression of truncated β subunits lacking the GBD with α and γ subunits yielded complexes that were active and normally regulated. However, coexpression of α and γ with full-length β caused accumulation of AMPK in large cytoplasmic inclusions that could be counterstained with anti-glycogen or anti-glycogen synthase antibodies. These inclusions were not affected by mutations that increased or abolished the kinase activity and were not observed by using truncated β subunits lacking the GBD. Our results suggest that the GBD binds glycogen and can lead to abnormal glycogen-containing inclusions when the kinase is overexpressed. These may be related to the abnormal glycogen storage bodies seen in heart disease patients with γ2 mutations.

AMPK β Subunit Targets Metabolic Stress Sensing to Glycogen

AMP-activated protein kinase (AMPK) is a multisubstrate enzyme activated by increases in AMP during metabolic stress caused by exercise, hypoxia, lack of cell nutrients [1], as well as hormones, including adiponectin and leptin [2, 3]. Furthermore, metformin and rosiglitazone, frontline drugs used for the treatment of type II diabetes, activate AMPK [4]. Mammalian AMPK is an αβγ heterotrimer with multiple isoforms of each subunit comprising α1, α2, β1, β2, γ1, γ2, and γ3, which have varying tissue and subcellular expression [5, 6]. Mutations in the AMPK γ subunit cause glycogen storage disease in humans [7], but the molecular relationship between glycogen and the AMPK/Snf1p kinase subfamily has not been apparent. We show that the AMPK β subunit contains a functional glycogen binding domain (β-GBD) that is most closely related to isoamylase domains found in glycogen and starch branching enzymes. Mutation of key glycogen binding residues, predicted by molecular modeling, completely abolished β-GBD binding to glycogen. AMPK binds to glycogen but retains full activity. Overexpressed AMPK β1 localized to specific mammalian subcellular structures that corresponded with the expression pattern of glycogen phosphorylase. Glycogen binding provides an architectural link between AMPK and a major cellular energy store and juxtaposes AMPK to glycogen bound phosphatases.

Two new mutations in the 3' coding region of the glycogen debranching enzyme in a glycogen storage disease type IIIa Ashkenazi Jewish patient.

Glycogen storage disease type III (GSD III) is an autosomal recessive disease caused by the deficiency of glycogen debranching enzyme (AGL). We report the finding of two new mutations in a GSD IIIa Ashkenazi Jewish patient. Both mutations are insertion of an adenine into a stretch of 8 adenines towards the 3' end of the coding region, one at position 3904 (3904insA) in exon 30, the second at position 4214 (4214insA) in exon 32. The mutations cause frameshifts and premature terminations of the glycogen debranching enzyme, the first causing a frameshift at amino acid 1304, the second causing a frameshift at amino acid 1408 of the total of 1532. These mutations demonstrate the importance of the 125 amino acids at the carboxy-terminus of the debrancher enzyme for its activity and support the suggestion that the putative glycogen binding domain is located in the carboxy-terminus of the AGL. The mutations cause distinctive single-strand conformation polymorphism (SSCP) patterns enabling easy detection.

The glycogen‐binding subunit of protein phosphatase‐1g from rabbit skeletal muscle

The glycogen-bound form of protein phosphatase-1 (PP-1G) was previously purified as a heterodimer composed of a 37-kDa catalytic (C) subunit and a proteolytically sensitive 103-kDa glycogen-binding (G) subunit [Stråhlfors, P., Hiraga, A. & Cohen, P. (1985) Eur. J. Biochem. 149, 295-303]. In this paper we demonstrate by a variety of criteria that the intact G subunit is a 161-kDa protein, and that the 103-kDa species (now termed G') is itself a product of proteolysis. A second phosphorylation site for cAMP-dependent protein kinase (termed site 2) was identified on the G subunit. The site 2 serine was phosphorylated at a comparable rate to site 1, and near stoichiometric phosphorylation could be achieved in the presence and absence of glycogen. Site 2 was dephosphorylated by PP-1 at a slow rate, whereas site 1 was resistant to autodephosphorylation. PP-1G, as well as the proteolytic activity responsible for degradation of the G subunit, remained tightly associated with glycogen-protein particles during washing with a variety of solvents. The PP-1G holoenzyme was released from glycogen-protein particles by dilution, with a dissociation half point corresponding to about 10 nM PP-1G. Binding experiments with purified PP-1G and glycogen indicated a bimolecular process with Kapp values corresponding to about 8 nM glycogen and 4 nM PP-1G. Binding was not significantly affected by increasing ionic strength to 0.5 M or variation of pH from 6 to 8. The results are consistent with a high-affinity glycogen-binding domain on the G subunit, and indicate that a physiological concentrations of phosphatase and glycogen, PP-1G should be almost entirely bound to glycogen.