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Previous Article | Next Article 
Molecular and Cellular Biology, January 2004, p. 352-361, Vol. 24, No. 1
0270-7306/04/$08.00+0 DOI: 10.1128/MCB.24.1.352-361.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.
Department of Genetics and Development, Columbia University, New York, New York 10032,1 St. Vincent's Institute of Medical Research, Fitzroy 3065, Victoria, Australia2
Received 28 July 2003/ Returned for modification 18 September 2003/ Accepted 3 October 2003
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ABSTRACT |
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INTRODUCTION |
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Snf1 and AMPK are heterotrimeric kinases with multiple subunit isoforms. Snf1 kinase comprises the catalytic subunit Snf1, a ß subunit (Gal83, Sip1, or Sip2), and the regulatory subunit Snf4. Snf1 kinase containing the Gal83 ß subunit will be referred to here as Snf1/Gal83 kinase. For AMPK, the corresponding subunit isoforms are designated 
1, 2, ß1, ß2, 
1, 2, and 3. Individual ß subunits of Snf1 and AMPK have distinct subcellular localizations (44, 47) and thereby presumably regulate access of the kinase to substrates. The Gal83 ß subunit is known to mediate interaction of the kinase with Sip4, a transcriptional activator of gluconeogenic genes that is dependent on Snf1 kinase for its function (27, 38, 42).
Recent studies identified a glycogen-binding domain in the AMPK ß1 subunit that is related to isoamylase domains found in glycogen and starch branching enzymes (21, 34). Mutation of conserved residues abolishes binding to glycogen in vitro (34). In mammalian cells, AMPK ß1 colocalizes with glycogen phosphorylase (34) and glycogen synthase (21); with the caveat that AMPK ß1 was overexpressed, these results suggest that AMPK binds to glycogen. Addition of glycogen does not affect AMPK catalytic activity in vitro (34). Previous biochemical and genetic evidence has implicated AMPK in regulation of glycogen metabolism, which in mammalian systems is under complex control by hormones and nutritional signals (36). Mutations of the subunit affect glycogen storage in pigs (5, 30) and cause a glycogen storage disease associated with cardiac abnormalities in humans (1). However, the physiological roles of the glycogen-binding domain of the ß1 subunit in AMPK function and glycogen metabolism remain unclear.
Glycogen is also an important storage carbohydrate in yeast (for review, see reference 11). Levels are low during exponential growth on glucose and rise rapidly before the onset of stationary phase, and the accumulated glycogen is then degraded slowly during prolonged starvation. Snf1 is one of several kinases that regulate glycogen metabolism, and Snf1 is required for glycogen accumulation (3, 19, 20, 41), regulation of the expression and catalytic activity of glycogen synthase (15, 49), and maintenance of glycogen stores (46).
We have taken advantage of yeast genetics to investigate the roles of the glycogen-binding domains of the ß subunits in vivo. The sequence of the glycogen-binding domain is conserved in two of the yeast ß subunits, Sip2 and Gal83. We found that Gal83 binds glycogen strongly in vitro, whereas Sip2 binds very weakly. We mutated conserved residues that are essential for glycogen binding of AMPK ß1 (34) and also analyzed another mutation that maps to a conserved residue (G235) within the glycogen-binding domain of Gal83 (9, 28). Alteration of Gal83 abolished glycogen binding in vitro and caused diverse phenotypes in vivo, positively affecting not only accumulation of glycogen but also transcriptional regulation. Surprisingly, the transcriptional regulatory phenotypes did not depend on the presence of glycogen within the cell, and the differences between the mutant and wild-type Gal83 were manifest even in a mutant strain (gsy1
gsy2) lacking glycogen synthase. Thus, mutation of the glycogen-binding domain positively affects Snf1/Gal83 kinase function by a mechanism that is independent of glycogen binding.
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MATERIALS AND METHODS |
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::TRP1 (42) and sip1::kanMX6 (44) have been described before, and the sip2::kanMX4 allele was amplified by PCR from strain BY24574. MCY4101 was derived from strains BY4741, BY15694, and BY15167 by genetic crossing, and the genotype was verified by using PCR. MCY4622 and MCY4626 are derivatives of 
1278b; the gal83 allele has been described elsewhere (45). Rich medium was yeast extract-peptone-dextrose, and synthetic (SD) and synthetic complete (SC) media contained appropriate supplementation to maintain selection for plasmids (37).
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700 bp) and the native terminator (500 bp). Proteins were tagged at the C terminus with green fluorescent protein (GFP). To introduce the R214Q mutation into pRT12, which expresses Gal83-GFP (44), PCR was performed using pRT12 as a template. Oligonucleotide Gal831A (5'-CCGATCTCGTAGGATTTGGG-3') and oligonucleotide Gal831B (5'-CTGAATCTTAACTCATTGTCAACAATAAACTGGAAACGATGAGTACCTGGAGGC-3') were used to generate a mutated fragment corresponding to nucleotides -119 to +671. Letters in bold indicate altered nucleotides. Oligonucleotides Gal832A (5'-GCCTCCAGGTACTCATCGTTTCCAGTTTATTGTTGACAATGAGTTAAGA-3') and Gal832B (5'-CAGTATTTGGGTCACGTATTTTG-3') were used to generate a mutated fragment corresponding to nucleotides 618 to 1233. These two fragments were cotransformed into yeast with pRT12 gapped with XcmI and NarI. The recombinant plasmid pHW33 was recovered in Escherichia coli and sequenced. We next introduced the W184A mutation by PCRs using pHW33 as a template. Oligonucleotide Gal831C (5'-CATAAGTCCAGGCTGTCCAGGGACTGGTACTAACCCGATCATCTTTCTCGCTCCGTAAAAGACCCAGT-3') and oligonucleotide Gal831A were used to generate a mutated fragment from nucleotides -119 to 600. Oligonucleotides Gal832C (5'-GGGGGGTAATAAAGTGTACGTTACTGGGTCTTTTACGGGAGCGAGAAAGATGATCGGGTTAGTACC-3') and Gal832B were used to generate a mutated fragment from nucleotides 510 to 1233 of the GAL83-R214Q sequence. These two fragments were used as described above to generate pHW39. pRT13, pHW40, and pHW41 are identical to pRT12, pHW39, and pOV81, respectively, except that the vector is pRS315 (39).
pHW30 is identical to pRT9, which expresses Sip2-GFP (44), except that the vector is pRS316 (39). To introduce the R216Q mutation, a mutagenic PCR was performed using pRT9 as a template. OL65 and oligonucleotide Sip21B (5'-CTCTAAGCTCATTATCCACTATAAACTGGAATCTATGTGTGCCTGGAAGC-3') were used to generate a mutated fragment corresponding to nucleotides -711 to 673. Oligonucleotide Sip22A (5'-CTAAGGCTGCTTCCAGGCACACATAGATTCCAGTTTATAGTGGATAATGAGCTTAGAG-3') and oligonucleotide Sip22B (5'-CGAACGATGGAGGCTACACAAAGTG-3') were used to generate a mutated fragment corresponding to nucleotides 615 to 1193. These two fragments were cotransformed into yeast with pHW30 gapped with MluI and BglII. pHW35 was recovered in E. coli and sequenced. We then introduced the W186A mutation by PCR using pHW35 as a template. OL65 and oligonucleotide Sip21C (5'-GCCATTATTGTCAGAATCAGGTATCAAACCGATCATTTTCCTCGCTTTGGTTGAATGAGCCTGTCAC-3') were used to generate a mutated fragment spanning nucleotides -711 to 590. Oligonucleotides Sip22C (5'-GGTGGTTCAAAAGTTTACGTGACAGGCTCATTCACCAAAGCGAGGAAAATGATCGGTTTCAT-3') and Sip22B were used to generate a mutated fragment corresponding to nucleotides 517 to 1193 of the SIP2-R216Q sequence. These two fragments were used as described above to generate pHW38. pOV81 is a derivative of pRT12 containing the GAL83-G235R sequence from pRJ324 (23).
To express the glycogen-binding domains of Gal83 and Sip2 in bacteria, DNA sequences corresponding to codons 152 to 251 and 154 to 253, respectively, were obtained by PCR using pRT12, pHW30, pHW38, pHW39, and pOV81 as templates. The amplified DNAs were cloned into the EcoRI/HindIII sites of the pProEX HT vector (Invitrogen), and clones were confirmed by sequencing.
Bacterial expression of the glycogen-binding domains of Gal83 and Sip2. Proteins corresponding to the glycogen-binding domains were expressed as His6 fusion proteins in BL21 cells. Cultures were grown to an optical density at 600 nm (OD600) of 0.6, and protein expression was induced with 1 mM isopropyl-ß-D-thiogalactopyranoside for 3 h at 37°C. Cells were harvested by centrifugation and lysed in phosphate-buffered saline (PBS) using an Emulsiflex-C5 high-pressure homogenizer (Avestin). Lysates were clarified by centrifugation and chromatographed on Ni-agarose. Nonspecific proteins were removed by washing the Ni-agarose with PBS containing 20 mM imidazole. Gal83 and Sip2 proteins were eluted with PBS containing 300 mM imidazole, concentrated to 0.5 ml, and purified by gel filtration on G-75 Sephadex.
Assay of glycogen binding. In vitro glycogen-binding assays were performed as described previously (34). Briefly, purified glycogen-binding domains of Gal83 or Sip2 (5 µg each) were incubated with 0.4% (wt/vol) bovine liver glycogen (type IX; Sigma) in a total volume of 0.25 ml in PBS and 0.5% Triton X-100 for 30 min at 4°C with mixing, followed by centrifugation at 200,000 x g for 60 min at 4°C. The supernatant was removed, and the pellet was briefly washed with 500 µl of PBS prior to resuspension to the original volume (0.25 ml). Gal83- or Sip2-containing fractions were identified by electrophoresis of proteins in Tris-tricine gels and immunoblotting with an anti-His6 tag antibody (Rockland Immunochemicals).
Assay of glycogen content. Glycogen assays were performed as described previously (33) with minor modifications. Mid-log cultures grown in selective SD plus 2% glucose were used to inoculate 25 to 100 ml of selective SD plus 2% glucose to an OD600 of 0.06. During growth of the culture, aliquots of cells (0.5 to 10 ml) were harvested by centrifugation and stored at -70°C. Cells were then resuspended in 25 µl of 0.25 M sodium carbonate, covered with 35 µl of mineral oil, and placed in a heating block at 95°C for 4 h. Fifteen microliters of 1 M acetic acid was added to neutralize the base, and then 60 µl of 0.2 M sodium acetate, pH 5.2, was added. Aspergillus niger amyloglucosidase (catalog number A-7420; Sigma) was added to a final concentration of 6.7 U/ml, and the mixture was incubated at 57°C for 12 h. The amount of glucose released was quantified using the glucose oxidase reaction (13).
Immunoblot analysis. Protein extracts were prepared for immunoblot analysis as described elsewhere (42). Proteins were separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis in 8% polyacrylamide and detected with anti-GFP antibody (Invitrogen) and ECL Plus reagents (Amersham).
Microscopy.
Cells were grown to mid-log phase in SD plus 2% glucose. Nuclei were stained by addition of 4',6-diamidino-2-phenylindole (DAPI) (0.8 µg/ml) for 5 min. Cells were collected by centrifugation and resuspended in 20 µl of residual medium. A 1.4-µl aliquot of the cell suspension was placed on a microscope slide. Cells were viewed using a Nikon Eclipse E800 fluorescence microscope. Images were taken with an Orca 100 camera (Hamamatsu) using Open Lab software (Improvision) and were processed in Adobe PhotoShop 5.5.
Kinase assays.
Immunoprecipitation and kinase assays were performed as described previously (26), except that cultures were grown selectively in SD plus 2% glucose. Polyclonal anti-LexA antibody was purchased from Invitrogen. For assays of kinase activity by phosphorylation of the SAMS peptide (HMRSA MSGLHLVKRR) substrate, cells were grown in SC plus 2% glucose to an OD600 of 0.7, collected by filtration, incubated in SC plus 0.5% glucose-2% glycerol-2% ethanol for 30 min, and then collected by filtration. Extracts were prepared and assays were performed as described previously (18). Briefly, Snf1 kinase was partially purified by chromatography on DEAE-Sepharose (Amersham Biosciences), and pooled fractions were assayed in triplicate for phosphorylation of the SAMS peptide in the presence of [-32P]ATP (8).
ß-Galactosidase assays. Transformants were grown to mid-log phase in selective SC or SD plus 2% glucose and were shifted to SC or SD plus 2% glycerol plus 2% ethanol for the time indicated. ß-Galactosidase activity was assayed in permeabilized cells and is expressed in Miller units (31).
Preparation of RNA and Northern blot analysis. Cells (50 ml) were collected by filtration, resuspended in 0.7 ml of TES (10 mM Tris-HCl [pH 7.5], 10 mM EDTA, 0.5% SDS), and frozen in liquid nitrogen. An equal volume of acid phenol was added to the sample, and cells were incubated at 65°C for 1.5 h with vortexing for 30 s 10 times at 3-min intervals and then for 30 s every 10 min. Samples were extracted four times with an equal volume of phenol and then twice with chloroform. RNA was precipitated with ethanol and resuspended in water. RNAs (40 µg) were separated by electrophoresis on a 1.2% agarose-morpholinepropanesulfonic acid gel containing formaldehyde and were transferred to a Hybond N+ membrane (Amersham Pharmacia Biotech). Probes were 32P-labeled using Ready-To-Go DNA labeling beads (Amersham Biosciences). The U3 small nucleolar RNA, encoded by SNR17A and SNR17B, was used as a loading control. GSY1 and GSY2 probes encompassed nucleotides 77 to 675 of the coding sequence.
Invasive growth assay. Transformants were streaked for single colonies on selective SC plus 2% glucose. Single colonies were resuspended in 100 µl of water, and 5 µl was spotted onto a yeast extract-peptone-dextrose plate. After 2 days at 30°C, plates were photographed, then placed under a gentle stream of tap water, rubbed with a gloved finger, and photographed again.
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RESULTS |
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gal83 strain on glycerol (data not shown).
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Double sip2 gal83 deletion reduces glycogen accumulation in vivo.
To assess the roles of the ß subunits in glycogen storage, we examined a sip2 gal83 strain expressing Sip2 and Gal83, separately and together. Cultures of all transformants showed identical growth kinetics in selective SD plus 2% glucose (Fig. 2A), and accumulation of glycogen was assayed during growth of the cultures (Fig. 2B). The sip2 gal83 double mutant had significantly diminished glycogen levels relative to transformants expressing both Sip2 and Gal83. Gal83 alone conferred normal glycogen accumulation, but transformants expressing only Sip2 may have a minor defect (Fig. 2B). Thus, both the Gal83 and Sip2 ß subunits contribute to Snf1 kinase function in promoting glycogen accumulation.
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gal83 strain expressing wild-type and mutant Gal83 and Sip2. Transformants expressing Gal83W184A,R214Q contained about twofold more glycogen than those expressing Gal83 at all phases of growth, independent of the presence of Sip2 (Fig. 2C and D). Gal83G235R caused similarly elevated glycogen levels (Fig. 2D). Levels were higher even during early exponential growth (9 h); values for Gal83, Gal83W184A,R214Q, and Gal83G235R were (mean ± standard error) 4.5 ± 0.6, 11.4 ± 2.5, and 12.4 ± 3.0 µg of glycogen/108 cells, respectively (Fig. 2D). These results indicate that mutation of critical residues in the glycogen-binding domain of Gal83 enhance glycogen accumulation. Protein levels, nucleocytoplasmic distribution, and Snf1 catalytic activity are normal for Gal83W184A,R214Q. To assess the possibility that the elevated glycogen levels simply reflect elevated levels of the mutant protein, we carried out immunoblot analysis, but we detected no difference (Fig. 3A). Another possibility is that these mutations alter the subcellular localization of Gal83, hence affecting the proximity of Snf1 kinase to particular substrates. GFP-tagged Gal83 is localized in the cytoplasm and excluded from the nucleus when cells are growing on glucose, but upon a shift to low glucose or a nonfermentable carbon source, Gal83 rapidly accumulates in the nucleus (44). GFP-tagged Gal83W184A,R214Q exhibited the same nucleocytoplasmic distribution (Fig. 3B). We cannot exclude differential association with glycogen, as glycogen granules in yeast cells can be resolved only by electron microscopy (6).
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cells expressing Gal83 or Gal83W184A,R214Q and LexA-tagged Snf1. Snf1 kinase was immunoprecipitated with anti-LexA and incubated with [-32P]ATP. Mutation of Gal83 did not increase the phosphorylation of Snf1 or Gal83 (Fig. 3C). The catalytically inactive LexA-Snf1T210A served as a control to confirm that phosphorylation reflected Snf1 activity.
We also assayed Snf1/Gal83 activity by phosphorylation of the SAMS peptide substrate (8). Snf1 kinase was partially purified from sip1 sip2 gal83 mutant cells (MCY4099) expressing Gal83 or Gal83W184A,R214Q and was incubated with SAMS peptide in the presence of [-32P]ATP. Incorporation of radiolabeled phosphate into the peptide was not significantly different (2.6 ± 0.6 and 1.8 ± 0.3 nmol/min/mg for Gal83 and Gal83W184A,R214Q, respectively, and 0.1 for the vector control; values are means of determinations for duplicate extracts). Thus, no alteration of catalytic activity was detected in vitro.
Gal83W184A,R214Q elevates expression of RNAs encoding glycogen synthase.
Previous work implicating Gal83 in transcriptional control (38, 42, 45) raised the possibility that increased transcription of key glycogen biosynthetic genes might be responsible for the increased glycogen accumulation. We examined levels of the GSY1 and GSY2 RNAs, which encode the two isoforms of glycogen synthase (10), in a gal83 mutant expressing Gal83W184A,R214Q or Gal83. Northern blot analysis showed that the W184A,R214Q mutation caused a twofold increase in the level of both GSY RNAs relative to a control RNA (Fig. 4); note that levels of the loading control RNA were lower in the mutant sample. Thus, an increase in expression of glycogen synthase likely contributes to the increase in glycogen accumulation.
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mutant is impaired in haploid invasive growth (45), a cellular response to carbon stress (7), at least in part due to effects on Snf1 kinase-dependent transcription of the FLO11 adhesin gene (25, 45). We expressed Gal83, Gal83W184A,R214Q, or Gal83G235R in a gal83 mutant of the invasive strain 1278b. The mutant proteins caused increased invasiveness (Fig. 5). This phenotype is dominant, as it was also observed in a GAL83 strain (Fig. 5).
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mutant, whereas GAL83-G235R partially relieves glucose inhibition of Sip4 and also improves activation (42).
To address the possibility that Gal83W184A,R214Q similarly affects Sip4, we assayed the ability of LexA-Sip4 to activate transcription of a lacZ reporter with LexA-binding sites (42). LexA-Sip4 was expressed from the ADH1 promoter in a gal83 mutant expressing Gal83, Gal83W184A,R214Q, or Gal83G235R. During growth in glucose, cells expressing either mutant protein produced two- to threefold higher ß-galactosidase activity than those expressing wild-type Gal83 (Fig. 6A). Following a shift to inducing conditions (nonfermentable carbon source), ß-galactosidase activity increased substantially, and the mutant proteins continued to confer higher activity. The role of Gal83 in upregulating invasive growth is distinct from its role in Sip4 activity, because sip4 mutants are hyperinvasive (7).
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mutant (Fig. 6B), suggesting that Cat8, like Sip4 (42), is dependent on the Snf1/Gal83 form of the kinase. We assayed activation of the CSRE-lacZ reporter in a gal83 mutant expressing Gal83, Gal83W184A,R214Q, or Gal83G235R. All strains showed normal glucose repression of the reporter, as expected because expression of both activators from their native promoters is glucose repressed. When cells were shifted to glycerol plus ethanol to induce activation of the CSRE, both Gal83W184A,R214Q and Gal83G235R conferred higher activation than wild-type Gal83 (Fig. 6B). These results show that mutations in the Gal83 glycogen-binding domain upregulate the function of native, Snf1 kinase-dependent transcription factors.
Mutation of the glycogen-binding domain affects the Snf1/Gal83 pathway in gsy1 gsy2 cells lacking glycogen.
The diverse phenotypes caused by the glycogen-binding domain mutations and, in particular, their effects on Snf1-dependent transcription under different growth conditions, including conditions when cells contain very little glycogen, cannot easily be accommodated by models involving glycogen binding. We therefore considered the possibility that some of these phenotypes are caused not by the failure to bind glycogen but rather by some other property of Gal83 that is altered by these mutations and affects Snf1/Gal83 kinase function. To test this model, we constructed a gsy1 gsy2 double mutant, which lacks glycogen synthase and does not synthesize glycogen (10). We reasoned that if glycogen binding is the critical factor responsible for the different phenotypes, then the mutant and wild-type Gal83 proteins should behave the same in a strain lacking glycogen altogether. If, on the other hand, the mutant and wild-type Gal83 proteins differ with respect to some other property, then they should still cause different phenotypes even in the absence of glycogen.
We compared isogenic wild-type and gsy1 gsy2 strains expressing Gal83 or Gal83W184A,R214Q with respect to LexA-Sip4 function and activation of the CSRE. We used reporter assays similar to those described above, but this pair of strains has a different genetic background. Mutation of the glycogen-binding domain caused increased ß-galactosidase expression not only in the wild-type control strain but also in the glycogen-deficient strain (Fig. 7). Moreover, the effect is dominant, as both strains carried a wild-type chromosomal GAL83 allele. Thus, mutation of the glycogen-binding domain confers phenotypes even in the absence of glycogen, indicating that the effect on Snf1/Gal83 function occurs by a glycogen-independent mechanism.
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DISCUSSION |
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Unexpectedly, the transcriptional regulatory phenotypes tested were not dependent on the presence of glycogen in the cell. First, mutant phenotypes were observed under growth conditions when cells contained very little glycogen. More importantly, mutation of the glycogen-binding domain of Gal83 conferred the same increase in Sip4 activator function and activation of the CSRE in a gsy1 gsy2 mutant strain lacking glycogen synthase as in a wild-type strain. Thus, mutation of the glycogen-binding domain affects Snf1/Gal83 kinase function by a mechanism that is independent of glycogen binding.
How do mutations in the glycogen-binding domain affect Snf1/Gal83 kinase? There are a number of possible mechanisms that we consider unlikely. First, we detected no alteration in the nucleocytoplasmic distribution of mutant Gal83. Although modest quantitative changes could have escaped notice, it is difficult to imagine such changes accounting for increased transcription during growth in glucose, when most Gal83 is cytoplasmic, and also in nonfermentable carbon sources, when Gal83 is nuclear. Second, in vitro assays revealed no increase in Snf1/Gal83 catalytic activity, suggesting that these mutations do not cause a conformational change in the kinase that favors its activation or augments activity. Third, mutant Gal83 could interact better with the catalytic subunit, thus enhancing Snf1 kinase function in Gal83-dependent processes. However, the glycogen-binding domain appears distinct from the site(s) of interaction with the catalytic subunit. A region that interacts (called the KIS region) was localized by evidence that residues 149 to 350 or 198 to 417 sufficed for interaction, but Sip2 residues 152 to 248, which encompass the glycogen-binding domain, did not interact with Snf1 (23), suggesting that the interacting site lies C-terminal to the domain. Fourth, mutation of the glycogen-binding domain could affect the interaction of Gal83 with particular targets; however, Sip4 interacts with residues 336 to 417 (42). It remains possible that mutation of the glycogen-binding domain indirectly affects the conformation of adjacent regions.
A final, and interesting, possibility is that this domain binds some other molecule that is closely related to glycogen and serves a signaling function. Mutation of the domain would reduce or abolish binding of this unknown molecule. This model is particularly attractive, because the binding of such a molecule could modulate Snf1/Gal83 catalytic activity in vivo, thereby nicely accounting for the manifestation of mutant phenotypes in the glycogen-deficient strain. Such a molecule could dissociate from the kinase during purification and hence not affect catalytic activity in our in vitro assays.
The existence of a glycogen-independent regulatory mechanism does not preclude direct effects of glycogen binding on some functions of Snf1/Gal83 kinase. For example, the increased accumulation of glycogen caused by these mutations may in part reflect the loss of binding to glycogen (an idea that cannot be tested genetically). However, it is also possible that the increased glycogen accumulation largely reflects the increased expression of GSY1 and GSY2 RNAs, which may, like other transcriptional phenotypes examined here, be controlled by glycogen-independent mechanisms. Glycogen is an important reserve carbohydrate that accumulates during conditions of nutrient stress and is then mobilized during periods of starvation, and Snf1 kinase has multiple roles in regulating glycogen metabolism (11, 15, 19, 20, 46, 49); hence, one can easily imagine the utility of a glycogen-sensing mechanism in regulating Snf1 kinase and a physiological role for the glycogen-binding domain in targeting the kinase to glycogen. Nonetheless, our genetic analysis provided no evidence for serious anomalies in glycogen storage or mobilization, but rather revealed unanticipated transcriptional regulatory phenotypes reflecting activation of the Snf1/Gal83 pathway.
These findings support a role for the glycogen-binding domain in modulating Snf1/Gal83 kinase function. This regulatory mechanism unexpectedly proved independent of glycogen binding. The interesting possibility that this domain binds an unidentified signaling molecule, related to glycogen, merits further investigation.
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ACKNOWLEDGMENTS |
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This work was supported by National Institutes of Health grant GM34095 to M.C., by the NHMRC (D.S. and B.E.K), and by the National Heart Foundation and Australian Research Council (B.E.K.). D.S. is an NHMRC RD Wright Fellow, and B.E.K. is a senior principal NHMRC Fellow.
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FOOTNOTES |
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REFERENCES |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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2. Brent, R., and M. Ptashne. 1985. A eukaryotic transcriptional activator bearing the DNA specificity of a prokaryotic repressor. Cell 43:729-736.[CrossRef][Medline]
3. Cannon, J., J. Pringle, A. Fiechter, and M. Khalil. 1994. Characterization of glycogen-deficient glc mutants of Saccharomyces cerevisiae. Genetics 136:485-503.[Abstract]
4. Carlson, M. 1999. Glucose repression in yeast. Curr. Opin. Microbiol. 2:202-207.[CrossRef][Medline]
5. Ciobanu, D., J. Bastiaansen, M. Malek, J. Helm, J. Woollard, G. Plastow, and M. Rothschild. 2001. Evidence for new alleles in the protein kinase adenosine monophosphate-activated gamma(3)-subunit gene associated with low glycogen content in pig skeletal muscle and improved meat quality. Genetics 159:1151-1162.
6. Coulary, B., M. Aigle, and J. Schaffer. 2001. Evidence for glycogen structures associated with plasma membrane invaginations as visualized by freeze-substitution and the Thiery reaction in Saccharomyces cerevisiae. J. Electron Microsc. 50:133-137.
7. Cullen, P. J., and G. F. Sprague. 2000. Glucose depletion causes haploid invasive growth in yeast. Proc. Natl. Acad. Sci. USA 97:13619-13624.
8. Davies, S. P., D. Carling, and D. G. Hardie. 1989. Tissue distribution of the AMP-activated protein kinase, and lack of activation by cyclic-AMP-dependent protein kinase, studied using a specific and sensitive peptide assay. Eur. J. Biochem. 186:123-128.[Medline]
9. Erickson, J. R., and M. Johnston. 1993. Genetic and molecular characterization of GAL83: its interactions and similarities with other genes involved in glucose repression in Saccharomyces cerevisiae. Genetics 135:655-664.[Abstract]
10. Farkas, I., T. A. Hardy, M. G. Goebl, and P. J. Roach. 1991. Two glycogen synthase isoforms in Saccharomyces cerevisiae are coded by distinct genes that are differentially controlled. J. Biol. Chem. 266:15602-15607.
11. Francois, J., and J. L. Parrou. 2001. Reserve carbohydrates metabolism in the yeast Saccharomyces cerevisiae. FEMS Microbiol. Rev. 25:124-145.
12. Gancedo, J. M. 1998. Yeast carbon catabolite repression. Microbiol. Mol. Biol. Rev. 62:334-361.
13. Goldstein, A., and J. O. Lampen. 1975. ß-D-Fructofuranoside fructohydrolase from yeast. Methods Enzymol. 42C:504-511.
14. Hardie, D. G., D. Carling, and M. Carlson. 1998. The AMP-Activated/Snf1 protein kinase subfamily: metabolic sensors of the eukaryotic cell? Annu. Rev. Biochem. 67:821-855.[CrossRef][Medline]
15. Hardy, T. A., D. Huang, and P. J. Roach. 1994. Interactions between cAMP-dependent and SNF1 protein kinases in the control of glycogen accumulation in Saccharomyces cerevisiae. J. Biol. Chem. 269:27907-27913.
16. Hedges, D., M. Proft, and K. D. Entian. 1995. Cat8, a new zinc cluster-encoding gene necessary for derepression of gluconeogenic enzymes in the yeast Saccharomyces cerevisiae. Mol. Cell. Biol. 15:1915-1922.[Abstract]
17. Hiesinger, M., S. Roth, E. Meissner, and H. J. Schuller. 2001. Contribution of Cat8 and Sip4 to the transcriptional activation of yeast gluconeogenic genes by the carbon source-responsive elements. Curr. Genet. 39:68-76.[CrossRef][Medline]
18. Hong, S.-P., F. C. Leiper, A. Woods, D. Carling, and M. Carlson. 2003. Activation of yeast Snf1 and mammalian AMP-activated protein kinase by upstream kinases. Proc. Natl. Acad. Sci. USA 100:8839-8843.
19. Huang, D., K. T. Chun, M. G. Goebl, and P. J. Roach. 1996. Genetic interactions between REG1/HEX2 and GLC7, the gene encoding protein phosphatase type 1 catalytic subunit in Saccharomyces cerevisiae. Genetics 143:119-127.[Abstract]
20. Huang, D., I. Farkas, and P. J. Roach. 1996. Pho85p, a cyclin-dependent protein kinase, and the Snf1p protein kinase act antagonistically to control glycogen accumulation in Saccharomyces cerevisiae. Mol. Cell. Biol. 16:4357-4365.[Abstract]
21. Hudson, E., D. Pan, J. James, J. Lucocq, S. Hawley, K. Green, O. Baba, T. Terashima, and D. G. Hardie. 2003. A novel domain in AMP-activated protein kinase causes glycogen storage bodies similar to those seen in hereditary cardiac arrythmias. Curr. Biol. 13:861-866.[CrossRef][Medline]
22. Jiang, R., and M. Carlson. 1996. Glucose regulates protein interactions within the yeast SNF1 protein kinase complex. Genes Dev. 10:3105-3115.
23. Jiang, R., and M. Carlson. 1997. The Snf1 protein kinase and its activating subunit, Snf4, interact with distinct domains of the Sip1/Sip2/Gal83 component in the kinase complex. Mol. Cell. Biol. 17:2099-2106.[Abstract]
24. Kemp, B. E., D. Stapleton, D. Campbell, Z. Chen, S. Murthy, M. Walter, A. Gupta, J. Adams, F. Kastis, B. VanDenderen, I. G. Jennings, T. Iseli, B. J. Michell, and L. A. Witters. 2003. AMP-activated protein kinase, super metabolic regulator. Biochem. Soc. Trans. 13:162-168.
25. Kuchin, S., V. K. Vyas, and M. Carlson. 2002. Snf1 protein kinase and the repressors Nrg1 and Nrg2 regulate FLO11, haploid invasive growth, and diploid pseudohyphal differentiation. Mol. Cell. Biol. 22:3994-4000.
26. Kuchin, S., V. K. Vyas, E. Kanter, S.-P. Hong, and M. Carlson. 2003. Std1p (Msn3p) positively regulates the Snf1 kinase in Saccharomyces cerevisiae. Genetics 163:507-514.
27. Lesage, P., X. Yang, and M. Carlson. 1996. Yeast Snf1 protein kinase interacts with Sip4, a C6 zinc cluster transcriptional activator: a new role for Snf1 in the glucose response. Mol. Cell. Biol. 16:1921-1928.[Abstract]
28. Matsumoto, K., A. Toh-E, and Y. Oshima. 1981. Isolation and characterization of dominant mutations resistant to carbon catabolite repression. Mol. Cell. Biol. 1:19-32.
29. McCartney, R. R., and M. C. Schmidt. 2001. Regulation of Snf1 kinase. Activation requires phosphorylation of threonine 210 by an upstream kinase as well as a distinct step mediated by the Snf4 subunit. J. Biol. Chem. 276:36460-36466.
30. Milan, D., J. Jeon, C. Looft, V. Amarger, A. Robic, M. Thelander, C. Rogel-Gaillard, S. Paul, N. Iannuccelli, L. Rask, H. Ronne, K. Lundstrom, N. Reinsch, J. Gellin, E. Kalm, P. Roy, P. Chardon, and L. Andersson. 2000. A mutation in PRKAG3 associated with excess glycogen content in pig skeletal muscle. Science 288:1248-1251.
31. Miller, J. H. 1972. Experiments in molecular genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
32. Minokoshi, Y., Y. B. Kim, O. D. Peroni, L. G. Fryer, C. Muller, D. Carling, and B. B. Kahn. 2002. Leptin stimulates fatty-acid oxidation by activating AMP-activated protein kinase. Nature 415:339-343.[CrossRef][Medline]
33. Parrou, J. L., and J. Francois. 1997. A simplified procedure for a rapid and reliable assay of both glycogen and trehalose in whole yeast cells. Anal. Biochem. 248:186-188.[CrossRef][Medline]
34. Polekhina, G., A. Gupta, B. J. Michell, B. van Denderen, S. Murthy, S. C. Feil, I. G. Jennings, D. J. Campbell, L. A. Witters, M. W. Parker, B. E. Kemp, and D. Stapleton. 2003. AMPK ß-subunit targets metabolic stress-sensing to glycogen. Curr. Biol. 13:867-871.[CrossRef][Medline]
35. Rahner, A., A. Scholer, E. Martens, B. Gollwitzer, and H. J. Schuller. 1996. Dual influence of the yeast Cat1p (Snf1p) protein kinase on carbon source-dependent transcriptional activation of gluconeogenic genes by the regulatory gene, CAT8. Nucleic Acids Res. 24:2331-2337.
36. Roach, P. J. 2002. Glycogen and its metabolism. Curr. Mol. Med. 2:101-120.[CrossRef][Medline]
37. Rose, M. D., F. Winston, and P. Hieter. 1990. Methods in yeast genetics: a laboratory course manual. Cold Spring Harbor Laboratory Press, Plainview, N.Y.
38. Schmidt, M. C., and R. R. McCartney. 2000. ß-Subunits of Snf1 kinase are required for kinase function and substrate definition. EMBO J. 19:4936-4943.[CrossRef][Medline]
39. Sikorski, R. S., and P. Hieter. 1989. A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics 122:19-27.
40. Thompson, J. D., D. G. Higgins, and T. J. Gibson. 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22:4673-4680.
41. Thompson-Jaeger, S., J. Francois, J. P. Gaughran, and K. Tatchell. 1991. Deletion of SNF1 affects the nutrient response of yeast and resembles mutations which activate the adenylate cyclase pathway. Genetics 129:697-706.[Abstract]
42. Vincent, O., and M. Carlson. 1999. Gal83 mediates the interaction of the Snf1 kinase complex with the transcription activator Sip4. EMBO J. 18:6672-6681.[CrossRef][Medline]
43. Vincent, O., and M. Carlson. 1998. Sip4, a Snf1 kinase-dependent transcriptional activator, binds to the carbon source-responsive element of gluconeogenic genes. EMBO J. 17:7002-7008.[CrossRef][Medline]
44. Vincent, O., R. Townley, S. Kuchin, and M. Carlson. 2001. Subcellular localization of the Snf1 kinase is regulated by specific ß subunits and a novel glucose signaling mechanism. Genes Dev. 15:1104-1114.
45. Vyas, V. K., S. Kuchin, C. D. Berkey, and M. Carlson. 2003. Snf1 kinases with different ß-subunit isoforms play distinct roles in regulating haploid invasive growth. Mol. Cell. Biol. 23:1341-1348.
46. Wang, Z., W. A. Wilson, M. A. Fujino, and P. J. Roach. 2001. Antagonistic controls of autophagy and glycogen accumulation by Snf1p, the yeast homolog of AMP-activated protein kinase, and the cyclin-dependent kinase Pho85p. Mol. Cell. Biol. 21:5742-5752.
47. Warden, S. M., C. Richardson, J. O'Donnell, D. Stapleton, B. E. Kemp, and L. A. Witters. 2001. Post-translational modifications of the beta-1 subunit of AMP-activated protein kinase affect enzyme activity and cellular localization. Biochem. J. 354:275-283.[CrossRef][Medline]
48. Wilson, W., S. Hawley, and D. Hardie. 1996. Glucose repression/derepression in budding yeast: SNF1 protein kinase is activated by phosphorylation under derepressing conditions, and this correlates with a high AMP:ATP ratio. Curr. Biol. 6:1426-1434.[CrossRef][Medline]
49. Wilson, W. A., A. M. Mahernholz, and P. J. Roach. 1999. Substrate targeting of the yeast cyclin-dependent kinase Pho85 by the cyclin Pcl10p. Mol. Cell. Biol. 19:7020-7030.
50. Woods, A., M. R. Munday, J. Scott, X. Yang, M. Carlson, and D. Carling. 1994. Yeast SNF1 is functionally related to mammalian AMP-activated protein kinase and regulates acetyl-CoA carboxylase in vivo. J. Biol. Chem. 269:19509-19515.
51. Yamauchi, T., J. Kamon, Y. Minokoshi, Y. Ito, H. Waki, S. Uchida, M. Noda, S. Kita K. Ueki, K. Eto, Y. Akanuma, P. Froguel F. Foufelle, P. Ferre, D. Carling, S. Kimura, R. Nagai, B. B. Kahn, and T. Kadowaki. 2002. Adiponectin stimulates glucose utilization and fatty-acid oxidation by activating AMP-activated protein kinase. Nat. Med. 8:1288-1295.[CrossRef][Medline]
52. Zhou, G., R. Myers, Y. Li, Y. Chen, X. Shen, J. Fenyk-Melody, M. Wu, J. Ventre, T. Doebber, N. Fujii, N. Musi, M. F. Hirshman, L. J. Goodyear, and D. E. Moller. 2001. Role of AMP-activated protein kinase in mechanism of metformin action. J. Clin. Investig. 108:1167-1174.[CrossRef][Medline]
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