Potato StubSNF1 interacts with StubGAL83: a ... - Wiley Online Library

0 downloads 0 Views 445KB Size Report
570 Lo´ rá nt Lakatos et al. Figure 1. Yeast two-hybrid analysis and in vitro binding assay of StubSNF1, StubGAL83 and yeast SNF4 protein–protein interactions.
The Plant Journal (1999) 17(5), 569–574

SHORT COMMUNICATION

Potato StubSNF1 interacts with StubGAL83: a plant protein kinase complex with yeast and mammalian counterparts Lo´ra´nt Lakatos1, Mathieu Klein2,† Rainer Ho¨fgen2 and Zso´fia Ba´nfalvi1,* 1Agricultural Biotechnology Center, H-2101 Godollo, ¨ ¨ ¨ PO Box 411, Hungary, and 2Max-Planck-Institut fur Molekulare Pflanzenphysiologie, ¨ Karl-Liebknecht-Str. 25, 14476 Golm, Germany

Summary StubSNF1 is a potato cDNA that encodes a protein kinase similar to the yeast SNF1 gene involved in transcriptional regulation of glucose-repressible genes. The yeast SNF1 functions in a complex with GAL83/SIP1/SIP2 and SNF4 proteins. We have used StubSNF1 as bait in a yeast two-hybrid system to screen for potato cDNAs encoding proteins that bind to StubSNF1. Three overlapping cDNAs, two different in size, were isolated. DNA sequence analysis revealed that they were orthologues of the yeast GAL83/ SIP1/SIP2 genes and their mammalian counterparts, AMPK β-subunits. The direct interaction between the potato proteins StubGAL83 and StubSNF1 was shown by an in vitro binding assay. Southern and Northern hybridisations revealed that StubGAL83 exists in a low copy number in the potato genome and is highly (but organ-specifically) expressed in potato. In contrast, StubSNF1 possesses low transcript levels in each organ, except in flowers where high amounts of StubSNF1 mRNA could be detected. We demonstrate here that StubGAL83 can also interact with yeast SNF4 in a yeast two-hybrid system suggesting that plant SNF1 kinases may function in complexes similar to those detected in yeast and mammals. Introduction The SNF1 protein kinases are widely conserved in eukaryotes. The Saccharomyces cerevisiae SNF1 gene is a serine/ threonine protein kinase required for expression of glucose-repressible genes in response to glucose deprivation (Celenza and Carlson, 1986). SNF1 is also known to be involved in other functions such as glycogen accumulation, Received 5 November 1998; revised 8 January 1999; accepted 11 January 1999. *For correspondence (fax 136 28 430482; e-mail [email protected]). †Present address: Metanomics GmbH & Co KG aA, Tegeler Weg 33, 10589 Berlin, Germany.

© 1999 Blackwell Science Ltd

sporulation and peroxisome biogenesis (Simon et al., 1992; Thompson-Jaeger et al., 1991), being part of a general mechanism through which yeast cells respond to carbon source starvation by activating protective systems against different types of stresses (Alepuz et al., 1997). The mammalian orthologue of the yeast SNF1, the AMPactivated protein kinase (AMPK), is involved in cellular stress responses that cause ATP depletion. To restore the normal ATP level in cells, activated AMPK inhibits the enzymes involved in cholesterol- and fatty acid biosynthesis. In addition, AMPK promotes ATP production by stimulating catabolic pathways (reviewed in Hardie and Carling, 1997). SNF1 homologs have been cloned from a variety of plants including rye, tobacco, Arabidopsis, barley, rice, sugar-beet and potato. These plant genes are sequential and functional homologs of SNF1, since rye and tobacco genes can complement the snf1 mutation in yeast. Previous studies on yeast and mammals showed that the conserved SNF1 family protects cells from nutritional and environmental stresses, and that similar functions can be expected in plants (reviewed in Halford and Hardie, 1998). The regulatory function of SNF1 kinases in plants, as in yeast and mammals (reviewed in Leclerc et al., 1998; Trumbly, 1992), can occur at both the post-translational and transcriptional level. Nitrate reductase and 3-hydroxy3-methylglutaryl-CoA reductase (HMGR) can be inactivated via phosphorylation by a member of the SNF1 kinase family (Douglas et al., 1997). Recently, it has been found that antisense expression of PKIN1, an SNF1 homolog isolated from potato, leads to the decrease of sucrose synthase mRNA accumulation in leaves and tubers (Purcell et al., 1998). In yeast cells, SNF1 is complexed with other proteins, including the activator subunit SNF4 and a member of the GAL83/SIP1/SIP2 family. The GAL83/SIP1/SIP2 family anchors SNF1 and SNF4 into a complex via distinct domains and serves as an adaptor. Furthermore, it was suggested that different members of the GAL83/SIP1/SIP2 family may take part in mediating the interaction of the kinase complex with different substrate proteins by their different N-terminal parts (Jiang and Carlson, 1997). Mammalian homologs of SNF4 (γ-subunit) and GAL83/ SIP1/SIP2 gene family (called β-subunit) are associated with AMPK showing that these components of the kinase complex have counterparts in mammals (Gao et al., 1996). In plants, however, proteins interacting with SNF1 have as 569

570 Lo´ra´nt Lakatos et al.

Figure 1. Yeast two-hybrid analysis and in vitro binding assay of StubSNF1, StubGAL83 and yeast SNF4 protein–protein interactions. (a) Yeast two-hybrid interactions between StubSNF1, StubGAL83 (aa. 4–289), ∆StubGAL83 (aa. 174–289), yeast SNF4 (ySNF4; Fields and Song, 1989) and the cloning vectors pBD-Gal4 and pAD-Gal4. Filter lift assay of three independent transformants was developed overnight. β-galactosidase activities are in Miller units and are the average values of three independent transformants with standard errors of less than 15% of the mean. (b) In vitro binding of StubSNF1 to StubGAL83. StubSNF1 was expressed as GST-and StubGAL83 (aa. 25–289) as TRX fusion protein. GST-StubSNF1 and GST (2 µg) were re-immobilised on glutathione-Sepharose 4B beads and incubated with TRX-StubGAL83 (2 µg) and TRX (10 µg). After extensive washing, bound proteins were boiled in sample buffer, analysed by electrophoresis on 10% SDS gel and blotted to Hybond C filter. Detection was carried out against S-Tag using the S-Tag Western blot kit. Size markers indicated on the left are in kDa.

yet not been identified. Since this might be crucial towards understanding the function of SNF1 in plants, we have attempted to isolate and characterise proteins interacting with a plant SNF1 in the yeast two-hybrid system. Results and discussion

Isolation of an SNF1-interacting protein To identify protein(s) interacting with SNF1, a potato leaf epidermal fragment yeast two-hybrid cDNA library (Ehrhardt et al., 1997) was screened with StubSNF1, a potato SNF1 cDNA isolated in our laboratory (Lakatos and Ba´nfalvi, 1997). From a total of about 600 000 transformants tested, 24 His1 yeast colonies were isolated. Eight of them were proven to be blue in the β-galactosidase filter lift assay. After partial sequencing, five different cDNAs were identified. Three of these were overlapping and showed homology to the yeast GAL83/SIP1/SIP2 gene family. The longest cDNA, designated StubGAL83, was 1026 bp in size. The other two cDNAs (∆StubGAL83) were the same size (516 bp) and were 100% identical to the corresponding part of the StubGAL83. Filter lift assay was carried out to confirm the positive interaction between StubSNF1 and GAL83. cDNAs encoding StubSNF1 and StubGAL83 or ∆StubGAL83 were cloned into both pBD-Gal4 and pAD-Gal4 vectors. Co-transformation experiments were carried out as shown in Figure 1(a, lanes 1–7). Interactions could be detected when StubSNF1

was present in the bait or pray vector with StubGAL83 or ∆StubGAL83 provided in the opposite vector (Figure 1a, lanes 1–3). In contrast, interaction was not detected in the presence of either StubSNF1 or StubGAL83 and an empty vector (Figure 1a, lanes 4–7). To confirm the direct interaction between StubSNF1 and StubGAL83, bacterially expressed fusion proteins were used for an in vitro binding experiment. StubSNF1 was expressed as a GST fusion protein. Due to plasmid stability problems, to express StubGAL83 as a thioredoxin (TRX) fusion protein, the BglII-XhoI fragment of the StubGAL83 cDNA extending over the putative SNF1 binding site (aa. 25–289) was used in binding experiments. The isolated and re-immobilised GST-StubSNF1 retained the provided TRX-StubGAL83 protein, while in control experiments with recombinant proteins GST-StubSNF1 versus TRX, GST versus TRX-StubGAL83 and GST versus TRX, no retention was detected (Figure 1b). These results suggest that StubSNF1 binds directly to StubGAL83.

DNA sequence analysis of the StubGAL83 cDNA The full length StubGAL83 cDNA consists of 1071 nucleotides, the longest open reading frame encodes a putative protein of 289 amino acids. Database searching revealed that the protein encoded by the StubGAL83 is homologous to the yeast GAL83/SIP1/SIP2 proteins (Erickson and Johnston, 1993; Yang et al., 1992) to the FOG1 protein of © Blackwell Science Ltd, The Plant Journal, (1999), 17, 569–574

Potato StubSNF1 interacts with StubGAL83 571 Figure 2. Sequence conservation of GAL83 in different organisms. Alignment of the predicted amino acid sequences of StubGAL83 of S. tuberosum (AJ012215), Scgal83 of S. cerevisiae (SwissProt Q04739), Fog1 of K. lactis (SwissProt Q00995), Atgal83 of A. thaliana (SwissProt O23481), AMPK β2 of human (SwissProt O43741). Gaps were introduced to optimise the alignment. Conservative KIS and ASC domains are indicated by arrows. The asterisk shows position 174 of StubGAL83, the starting amino acid of the ∆StubGAL83 clone.

Kluveromyces lactis (Goffrini et al., 1996), to the mammalian AMPK β-subunits (Gao et al., 1996; Thornton et al., 1998), and to an Arabidopsis thaliana ORF (Bevan et al., 1996) encoding a 259 aa. protein (Figure 2). The highest homology was found at the Arabidopsis protein with 51% identity and 63% similarity. Searching in the plant EST databases showed that two different transcripts in Arabidopsis (accession no. N96609, AA394895) and one in rice (accession no. AA751769) were very similar to the StubGAL83 cDNA. The partial EST cDNA clone (accession © Blackwell Science Ltd, The Plant Journal, (1999), 17, 569–574

no. N96609) corresponds to the Arabidopsis ORF described by Bevan et al. (1996) while AA394895 is different. These data suggest that there are at least two StubGAL83 homologous genes in the Arabidopsis genome. In yeast GAL83/SIP1/SIP2 proteins and in the mammalian AMPK β-subunits, two distinct well-conserved domains were identified. The first internal domain, called the KIS domain (kinase association domain), is responsible for the interaction with SNF1. The C-terminal ASC (association with the SNF1 complex) domain interacts with SNF4 (Jiang

572 Lo´ra´nt Lakatos et al. and Carlson, 1997; Thornton et al., 1998). Sequence alignment revealed that StubGAL83 contains both of these wellconserved regions (Figure 2), suggesting that StubGAL83 might be involved in similar interactions described for its counterparts in yeast and mammals. The yeast two-hybrid screen resulted in the isolation of StubGAL83 (aa. 4–289) and ∆StubGAL83 clones (aa. 174– 289). ∆StubGAL83 contains only the last nine amino acids of the KIS domain as defined by Thornton et al. (1998). Nevertheless, based on quantitative determination of βgalactosidase activities, ∆StubGAL83 interacts with StubSNF1 as efficiently as StubGAL83 (32 and 34 Miller units, respectively; Figure 1a, lanes 1–2). Thus, the last nine amino acids of the KIS domain might be sufficient to mediate direct association between StubSNF1 and StubGAL83.

StubGAL83 interacts with yeast SNF4

Figure 3. Expression analysis and genome organisation of StubGAL83. (a) Comparison of the level of expression of StubSNF1 and StubGAL83 in different organs of potato. Each lane contains 10 µg of total RNA extracted from roots (R), stem (S), leaves (L), stolon (St), tuber (T), in vitro stolon (st), in vitro tuber (t), berry (B), and flower (F). The blot was hybridised to the indicated probes. Exposure time was 5 days in both cases. The ethidium bromide stained gel shows the approximate equal loading of total RNA in each lane. (b) Southern blot analysis of potato (S. tuberosum cv. Keszthelyi 855) DNA. 10 µg DNA was digested with EcoRI (E) and HindIII (H) and run on a 1% agarose gel then blotted onto a Hybond N1 filter and hybridised with the ∆StubGAL83. The positions of the size markers in kb are indicated.

The SNF1 complex in yeast is a heterotrimetric protein of three subunits, i.e. the catalytic subunit (SNF1 kinase), the adaptor (GAL83/SIP1/SIP2) and the activator subunit (SNF4; Jiang and Carlson, 1997). With the help of yeast two-hybrid screening we have identified the StubGAL83 protein that possibly corresponds to the adaptor subunit of the yeast SNF1 kinase complex. Furthermore, Abe et al. (1995) isolated a cDNA from Phaseolus vulgaris encoding a protein similar to the yeast SNF4. These findings led us to speculate that SNF1 in plants may also function as a heterotrimeric complex. To test this hypothesis, interactions of StubGAL83 and StubSNF1 with the yeast SNF4 were investigated in the yeast two-hybrid system. When StubGAL83 or ∆StubGAL83 were used as baits and yeast SNF4 as prey, interactions almost as strong as measured between StubSNF1 and StubGAL83 (22 and 25 Miller units versus 34) were detected (Figure 1a, lanes 8–9). However, when StubSNF1 was tested with the yeast SNF4, no interaction was found (Figure 1a, lane 11). This negative result does not exclude the possibility that the plant SNF1 protein kinase may function as a heterotrimeric complex similar to that found in yeast and mammals. Possible explanations are that the yeast SNF4 is unable to substitute the plant SNF4 in interaction with StubSNF1, or that there is no association between StubSNF1 and SNF4 proteins in the absence of the adaptor StubGAL83.

StubSNF1 mRNA were detected. The same filter was hybridised with StubGAL83. In contrast to StubSNF1, a high level of expression was found in most of the organs, especially in leaf and stem, but was very low in root and berry (Figure 3a). StubSNF1 belongs to the SnRK1a group of genes as classified by Halford and Hardie (1998). The equal levels of transcript in different organs is a common feature of this group of genes, suggesting post-transcriptional activation probably via phosphorylation in a way similar to that found with mammals in the case of AMPK. Regulation of StubGAL83, however, might be transcriptional. Organ-specific expression of mammalian GAL83 orthologues is already known. The human AMPK β2-subunit, for example, is actively transcribed in skeletal muscle and liver, in organs where the SNF1 complex is enzymatically functional, but not in the lung or kidney where no enzymatic activity could be detected (Thornton et al., 1998). Thus, we speculate that the highest expression level of StubGAL83 in leaves may be in correlation with its organ-specific role in targeting the SNF1 complex towards different substrates. Another possibility is that the SNF1 complex plays a major role in flower where both StubSNF1 and StubGAL83 are actively transcribed.

Expression of StubSNF1 and StubGAL83

Copy number of StubGAL83

Expression patterns of StubSNF1 and StubGAL83 were determined by Northern hybridisation in different organs of potato including root, shoot, leaf, stolon, tuber, in vitroinduced tuber and stolon, berry and flower. The transcript level of StubSNF1 was very low in all organs of potato except the flower, where high amounts of steady-state

To determine the copy number of the StubGAL83 gene in potato, Southern blot analysis was carried out using the ∆StubGAL83 as a probe. According to the autoradiogram shown in Figure 3(b) the copy number of StubGAL83 in potato is low or there is only a little variation within the gene family, as was suggested for SNF1-related genes in © Blackwell Science Ltd, The Plant Journal, (1999), 17, 569–574

Potato StubSNF1 interacts with StubGAL83 573 potato by Man et al. (1997). The sequence identity and similarity between the protein encoded by PKIN1 and StubSNF1 are 66% and 72%, respectively. Apart from StubSNF1, a cDNA fragment 98% identical to PKIN1 was also isolated from the S. tuberosum cv. Keszthelyi 855 in our laboratory (L. Lakatos and Z. Ba´nfalvi, unpublished results). Thus, there are at least two different members of the SNF1-related genes in potato that may interact with different proteins and participate in specific signal transduction or regulatory pathways. Since in yeast the GAL83/ SIP1/SIP2 family mediate the association of SNF1 with specific substrates and target the kinase to specific intracellular locations (Jiang and Carlson, 1997), we hope that the isolation and characterisation of StubGAL83 presented here will facilitate the understanding of function of the SNF1 complex in plant cells.

Experimental procedures

Plant material Potato cv. Keszthelyi 855 was grown and in vitro microtubers were induced as described by Ba´nfalvi et al. (1996).

library in vector pAD-Gal4. The library was established from epidermal fragments of S. tuberosum cv. De´sire´e leaves as described by Ehrhardt et al. (1997). After 4–6 days of incubation, His1 colonies were screened for β-galactosidase activity using a filter lift assay (Jiang and Carlson, 1996). Quantification of βgalactosidase activity was as described by Miller (1972). Positive colonies were picked from SDG-Trp-Leu-His plates and grown overnight in SDG-Trp-Leu liquid medium. DNA prepared from these cultures was transformed into DH5α and selected for ampicillin resistance bearing the AD library plasmid.

Isolation of the 59-end of the StubGAL83 cDNA Isolation of the 59-end of StubGAL83 cDNA was performed by two rounds of nested PCR using the entire yeast two-hybrid cDNA library as a template. Primer GALT (position 168–187 on the resulted full length StubGAL83) 59-GAC CAT CAA GTC AGC CGA AG was used for the first round PCR, and GALK (position 139–158 on the resulted full length cDNA StubGAL83) 59-AGG TCA GAG CGT GGT GAT CT for the second PCR reaction as gene-specific primers. For a non-specific primer, Gal4AD primer 59-TTC GAT GAT GAA GAT ACC was used. After the second PCR reaction, the longest PCR product was cloned into an EcoRV-digested pBluescript and sequenced using the USB kit Sequenase version 2.0 and partly by the company MWG-Biotech, Germany. Full length StubGAL83 was created by cloning the EcoRI-BglII fragment of the PCR product representing the missing 59 end into the cDNA clone StubGAL83 isolated in library screening.

Plasmid constructions and library screening Plasmid constructs were generated using standard methods (Sambrook et al., 1989). The protein coding region of the StubSNF1 cDNA (Lakatos and Ba´nfalvi, 1997) was amplified by PCR with the SNF1BAL primer 59-GGG AAT TCA TGG ACG GAA CAG CAG TGC AAG and the SNF1JOBB primer 59-TCA AAG TAC GCG AAG CTG AGC A. The PCR product was digested with EcoRI then ligated into an EcoRIEcoRV digested pBluescript (Stratagene) resulting in pLL31. DNA sequence of the insert was determined then cut with HindIII, filledin with Klenow, digested with EcoRI and ligated into the EcoRISmaI digested pBD-Gal4 vector (Stratagene), resulting in the bait vector expressing the Gal4BD-StubSNF1 fusion protein (pLL32). In order to express the StubSNF1 as a Gal4AD (Gal4 activation domain) fusion protein, the EcoRI-XbaI insert of the pLL32 was cloned into the pAD-Gal4 vector (Stratagene). To create plasmids expressing the StubGAL83 (aa. 4–289) and ∆StubGAL83 (aa. 174–289) as Gal4BD fusion proteins, the EcoRIXhoI insert of the cDNA clones isolated from the yeast two-hybrid screen were inserted into pBD-Gal4. The plasmid expressing the GST-StubSNF1 hybrid was created by inserting the HindIII filled-in, BamHI fragment of pLL31 into the BamHI-SmaI digested pGEX 2T (Pharmacia). To obtain a plasmid expressing the StubGAL83 (aa. 25–289) as a thioredoxin (TRX) fusion protein with an amino-terminal 6 3 HisTag, the BglII-XhoI fragment of the pStubGAL83 was cloned into the BamHI-XhoI cut pET32a1 (Novagen).

Library screening Yeast transformations were performed by the lithium acetate method described by Schiestl and Gietz (1989). Strain YRG-2 was transformed with pLL32 to Trp prototrophy. The resulting strain was used in the interaction trap screening with an oriented cDNA © Blackwell Science Ltd, The Plant Journal, (1999), 17, 569–574

Nucleic acid isolation and hybridisation Total RNAs were extracted as described by Stiekema et al. (1988). Hybridisation of filters were carried out in Church buffer (Church and Gilbert, 1984). Genomic DNA was isolated by the method of Shure et al. (1983). Hybridisation was performed in 50 mM Tris pH 7.5, 1 M NaCl, 1% SDS, 10% dextran sulfate, 100 µg ml–1 denatured salmon sperm DNA at 65°C overnight. Washing of the filter was carried out first in distilled water for 2 min at room temperature then in 2 3 SSC, 1% SDS for 30 min at 65°C.

Protein expression and in vitro binding assay Protein expression and binding assay was carried out by the method of Jiang and Carlson (1996) with the following modifications: (i) bacterial pellets were resuspended in cold ST (30 mM Tris pH 8.0, 120 mM NaCl) with 100 µg ml–1 lysosyme; (ii) the TRX fusions were eluted with ST containing 20, 50, 300, 500 mM imidazole; and (iii) Western blot analysis was carried out against S-Tag using the S-Tag Western blot kit (Novagen).

Aknowledgements We are grateful to Dr M. Carlson (Columbia University, NY, USA) for the plasmid pNI12 carrying the yeast SNF4 in the vector pADGal4 and to Dr T. Ehrhardt and Dr B. Mu¨ller-Ro¨ber for providing the pAD EF cDNA library. We thank X. Sztanko´ for technical assistance, G. Taka´cs for photos and J. Lloyd for critical reading of the manuscript. This work was supported by the VolkswagenStiftung I/71 757 and the Hungarian grant OTKA T 02932.

574 Lo´ra´nt Lakatos et al. References Abe, H., Kamiya, Y. and Sakurai, A. (1995) A cDNA clone encoding yeast SNF4-like protein (accession no. U40713) from Phaseolus vulgaris L. (PGR95–126). Plant Physiol. 110, 336. Alepuz, P.M., Cunningham, K.W. and Estruch, F. (1997) Glucose repression affects ion homeostasis in yeast through the regulation of the stress-activated ENA1 gene. Mol. Microbiol. 1, 91–98. Ba´nfalvi, Z., Molna´r, A., Molna´r, G., Lakatos, L. and Szabo´, L. (1996) Starch synthesis, and tuber storage protein genes are differently expressed in Solanum tuberosum and in Solanum brevidens. FEBS Lett. 383, 159–164. Bevan, M., Bancroft, I., Bent, E. et al. (1996) Analysis of 1.9 Mb of contiguous sequence from chromosome 4 of Arabidopsis thaliana. Nature, 391, 485–488. Celenza, J.L. and Carlson, M. (1986) A yeast gene that is essential for release from glucose repression encodes a protein kinase. Science, 233, 1175–1180. Church, G.M. and Gilbert, W. (1984) Genomic sequencing. Proc. Natl Acad. Sci. USA, 81, 1991–1995. Douglas, P., Pigaglio, E., Ferrer, A., Halford, N.G. and MacKintosh, C. (1997) Three spinach leaf nitrate reductase-3-hydroxy-3methylglutaryl-CoA reductase kinases that are required by reversible phosphorylation and/or Ca21 ions. Biochem. J. 325, 101–109. Ehrhardt, T., Zimmermann, S. and Mu¨ller-Ro¨ber, B. (1997) Association of plant K1in channels is mediated by conserved Ctermini and does not affect subunit assembly. FEBS Lett. 409, 166–170. Erickson, J.R. and Johnston, M. (1993) Genetic and molecular characterization of GAL83: its interaction and similarities with other genes involved in glucose repression in Saccharomyces cerevisiae. Genetics, 135, 655–664. Fields, S. and Song, O. (1989) A novel genetic system to detect protein–protein interactions. Nature, 340, 245–246. Gao, G., Fernandez, C.S., Stapleton, D., Auster, A.S., Widmer, J., Dyck, J.R., Kemp, B.E. and Witters, L.A. (1996) Non-catalytic beta- and gamma-subunit isoforms of the 59-AMP-activated protein kinase. J. Biol. Chem. 271, 8675–8681. Goffrini, P., Ficarelli, A., Donnini, C., Lodi, T., Puglisi, P.P. and Ferrero, I. (1996) FOG1 and FOG2 genes, required for the transcriptional activation of glucose-repressible genes of Kluyveromyces lactis, are homologous to GAL83 and SNF1 of Saccharomyces cerevisiae. Curr. Genet. 4, 316–326. Halford, N.G. and Hardie, D.G. (1998) SNF1-related protein kinases: global regulators of carbon metabolism in plants? Plant Mol. Biol. 37, 735–748. Hardie, D.G. and Carling, D. (1997) The AMP-activated protein kinase. Fuel gauge of the mammalian cell? Eur. J. Biochem. 246, 259–273. Jiang, R. and Carlson, M. (1996) Glucose regulates protein interactions within the yeast SNF1 protein kinase complex. Genes Dev. 10, 3105–3115.

Jiang, R. and Carlson, M. (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. Lakatos, L. and Ba´nfalvi, Z. (1997) Nucleotide sequence of a cDNA clone encoding an SNF1 protein kinase homologue (Accession no. U83797): from Solanum tuberosum (PGR97–043). Plant Physiol. 113, 1004. Leclerc, I., Kahn, A. and Doiron, B. (1998) The 59-AMP-activated protein kinase inhibits the transcriptional stimulation by glucose in liver cells, acting through the glucose response complex. FEBS Lett. 431, 180–184. Man, A.L., Purcell, P.C., Hannappel, U. and Halford, N.G. (1997) Potato SNF1-related protein kinase: molecular cloning, expression analysis and peptide kinase activity measurements. Plant Mol. Biol. 34, 31–43. Miller, J.H. (1972) Experiments in Molecular Genetics. Cold Spring Harbor: Cold Spring Harbor Laboratory Press. Purcell, P.C., Smith, A.M. and Halford, N.G. (1998) Antisense expression of a sucrose non-fermenting-1-related protein kinase sequence in potato results in decreased expression of sucrose synthase in tubers and loss of sucrose-inducibility of sucrose synthase transcripts in leaves. Plant J. 14, 195–202. Sambrook, J., Fritch, E.F. and Maniatis, T. (1989) Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor: Cold Spring Harbor Laboratory Press. Schiestl, R.H. and Gietz, R.D. (1989) High efficiency transformation of intact yeast cells using single stranded nucleic acids as a carrier. Curr. Genet. 5–6, 339–346. Shure, M., Wessler, S. and Fedoroff, N. (1983) Molecular identification and isolation of the Waxy locus in maize. Cell, 35, 225–233. Simon, M., Binder, M., Adam, G., Hartig, A. and Ruis, H. (1992) Control of peroxisome proliferation in Saccharomyces cerevisiae by ADR1, SNF1 (CAT1, CCR1) and SNF4 (CAT3). Yeast, 4, 303–309. Stiekema, W.J., Heidkamp, F., Dirkse, W.G., Van Beckum, J., De Haan, P., Bosh, T. and Lauwerse, J.D. (1988) Molecular cloning and analysis of four tuber specific mRNAs. Plant Mol. Biol. 11, 255–269. Thompson-Jaeger, S., Francois, J., Gaughran, J.P. and Tatchell, K. (1991) Deletion of SNF1 affects the nutrient response of yeast and resembles mutations which activate the adenylate cyclase pathway. Genetics, 29, 697–706. Thornton, C., Snowden, M.A. and Carling, D. (1998) Identification of a novel AMP-activated protein kinase beta subunit isoform that is highly expressed in skeletal muscle. J. Biol. Chem. 273, 443–12450. Trumbly, R.J. (1992) Glucose repression in the yeast Saccharomyces cerevisiae. Mol. Microbiol. 6, 15–21. Yang, X., Hubbard, E.J. and Carlson, M. (1992) A protein kinase substrate identified by the two-hybrid system. Science, 257, 680–682.

EMBL accession number AJ012215 (StubGAL83 cDNA).

© Blackwell Science Ltd, The Plant Journal, (1999), 17, 569–574