Characterization of the Hub1 protein modification system

• Applicant: Professor Dr. Gunnar Dittmar
• Subject Area: Cell Biology
• Term: from 2003 to 2006
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 5401172

I have found that the newly identified ubiquitin-like protein Hub1 acts as a posttranslational modifier. Two substrate proteins of the system have also been identified, Hbt1 and Sph1. Both proteins are posttranslational modified and are involved in the regulation of cell polarity. The HUB1 gene is not essential in S. cerevisiae, but the loss of this evolutionary extraordinarily conserved gene is lethal in higher organisms. S. cerevisiae cells lacking hub1 show deformed cell morphology and are more sensitive to high salt conditions. I propose to identify the enzymatic machinery necessary to form the covalent Hub1 substrate adducts. I anticipate that the enzymatic machinery consists of an activating and a conjugating component like for ther ubiquitin-like proteins. Covalent affinity chromatography will be used to isolate the activating protein. Alternatively a new suicide-reagent will be used that reacts with the active center of the enzyme. The conjugating component will be isolated either by covalent affinity chromatography or by screening a library of all 6200 ORF of S. cerevisiae expressed as GST-fusions. Hub1 is probably expressed as a precursor form that needs to be maturated by proteolytic cleavage. The identification of this enzyme using fluorescence based cleavage assay and its biochemical characterization is another aim of this proposal.

• DFG Programme: Research Grants