Activation of Gal4p by galactose-dependent interaction of galactokinase and Gal80p

Science

Volumn 272, Issue 5268, 1996, Pages 1662-1665

  Zenke, F T ^a   Engels, R ^a   Vollenbroich, V ^a   Meyer, J ^a   Hollenberg, C P ^a   Breunig, K D ^a  

^a HEINRICH HEINE UNIVERSITY   (Germany)

Author keywords

[No Author keywords available]

Indexed keywords

ADENOSINE TRIPHOSPHATE; GALACTOKINASE; GALACTOSE;

ARTICLE; CONTROLLED STUDY; ENZYME ACTIVATION; GENE ACTIVATION; NONHUMAN; PHOSPHORYLATION; PRIORITY JOURNAL; PROTEIN PROTEIN INTERACTION; SIGNAL TRANSDUCTION; TRANSCRIPTION REGULATION; YEAST;

EID: 0029972916     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.272.5268.1662     Document Type: Article

References (32)

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13. J. Meyer, thesis, Heinrich-Heine-Universität Düsseldorf (1993).
14. Whereas the gal1 mutant is noninducible, a gal1 gal80 double mutant has exactly the same constitutive phenotype as a gal80 mutant (9, 23).
15. - mutant on a multicopy plasmid (pEAG80His), the mutation was fully complemented as assayed by the expression of the Lac9p-controlled β-gaiactosidase gene.
16. J. Crowe et al., in Methodsin Molecular Biology, A. J. Harwood, Ed. (Humana, Totowa, NJ, 1990), pp. 371-387.
17. -) for growth on galactose [J. Meyer et al., in preparation].
18. β-Galactosidase is encoded by the LAC4 gene, which is one of the KlGal4p-controlled genes in K. lactis. Enzymatic activity is strictly controlled by transcriptional regulation.
19. A gal80::URA3 disruption was replaced by the ScGAL80 structural gene fused to the KlGAL80 promoter.
20. A cell extract from a gal1 gal80 deletion strain transformed with pEAG80His [compare (11)] was loaded on a Ni-NTA-agarose column (3 ml), washed with extraction buffer (50 mM Hepes buffered with NaOH to pH 8, 100 mM NaCl, 10% glycerol) containing 50 mM imidazole, and eluted with a 30-ml linear gradient of 50 to 200 mM imidazole.
21. 2, 7 mM β-mercaptoethanol, 10% glycerol).
22. 2-terminal end of klGal1p. The galactokinase activity of the fusion protein was comparable with that of the wildtype protein.
23. F. T. Zenke et al., data not shown.
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30. Protein extracts from yeast transformants grown in synthetic minimal medium (SM) with 3% glycerol were prepared by glass bead lysis in binding buffer. Extracts were incubated for 1 hour with equilibrated Ni-NTA-agarose at 4°C. Galactose (2%) and 2 mM ATP were added during the binding and washing steps. The resin was washed with binding buffer containing 50 mM imidazole and heated for 10 min at 95°C in SDS sample buffer, followed by fractionation of the proteins by SDS-polyacrylamide gel electrophoresis (SDS-PAGE).
31. F. T. Zenke, thesis, Heinrich-Heine-Universität Düsseldorf (1995).
32. We thank P. Kuger for expert technical assistance and G. Cardinali for supplying the ADHI-gal1-m1 fusion. Many colleagues, in particular J. Dohmen and R. Kölling, have contributed with helpful critical comments on the manuscript. Supported by Deutsche Forschungsgemeinschaft grant Br921 to K.D.B. and Bundesministerium für Bildung und Forschung-Schwerpunkt Biocatalysis to C.P.H.