Systematic identification and characterization of missing GEFs for GTPases

• Applicants: Professor Dr. Roger S. Goody; Professor Dr. Aymelt Itzen
• Subject Area: Biochemistry ; Cell Biology
• Term: from 2014 to 2018
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 258576031

The aim of the project is to develop and apply new methods for the identification and characterization of guanine nucleotide exchange factors (GEFs) that operate on Ras-superfamily GTPases. While a large number of GEFs has been assigned to some members of this family, relatively few are known for others. In the case of the Rab proteins, many of the more than 60 family members are not yet associated with known GEFs, even in cases that have been studied extensively over many years, such as Rab6. Two main approaches will be undertaken. In one of these, GTP/GDP depletion of cell lysates will be used to pull down cognate GEFs to specific GTPases using GTPase coated beads, after which treatment with GTP will specifically elute co-immobilized GEFs. Advantages of this approach are the GTP-specific elution and the possibility that other proteins associated with the GEFs, which are often multidomain proteins involved in numerous protein-protein interactions, will also be eluted, allowing the simultaneous identification of GEFs and interaction partners by mass spectrometry. In another approach, guanine nucleotides will be covalently bound to GTPase cysteine mutants, thus locking them irreversibly in their respective nucleotide states. As well as the active (GTP) and inactive (GDP) states, this includes GMP and guanosine bound states that function essentially as nucleotide free GTPases, interacting strongly with GEFs in a nucleotide-independent manner. These will be used in pull down experiments, again identifying bound proteins by mass spectrometry. Newly Identified GEFs and their interacting proteins will be extensively characterized both in vitro (3-D structure determination, kinetics) and in vivo using cell biological methods.

• DFG Programme: Research Grants
• International Connection: France
• Participating Person: Professor Dr. Bruno Goud