CLONING, ZN2-NUCLEOTIDE EXCHANGE FACTOR HUMAN MSS4( BINDING, AND STRUCTURAL CHARACTERIZATION OF THE GUANINE)

The Sec4/Ypt1/Rab family of small GTP-binding proteins are involved in the regulation of intracellular vesicular transport. A rat gene, mss4 , that encodes a guanine nucleotide exchange factor (GEF) for Sec4 was recently cloned by its ability to rescue defects in protein transport of a yeast temperature-sensitive (ts) mutant, sec4-8. We describe her ein the cloning, bacterial expression, and biochemical characterizatio n of human Mss4. As expected, both the cDNA and its encoded amino acid sequences are highly conserved between the human and rat mss4. Solubl e and functional Mss4 protein was obtained by expressing the gene as a glutathione-S-transferase fusion protein in Escherichia coli. Subsequ ent biochemical analysis revealed that Mss4 binds 1 equiv of Zn2+, and zinc is essential for the stability of the protein. Utilizing multidi mensional heteronuclear NMR techniques, we assigned most of the H-1, N -15, and C-13 resonances of this 14-kDa protein. Its secondary structu re was also deduced from slowly exchanging amide protons, characterist ic NOEs, and (3)J(NH-C alpha H) coupling constants. The protein contai ns a central seven-stranded antiparallel beta sheet, flanked by two sm all beta sheets. Many resonances pertaining to a loop region of the mo lecule cannot be identified, suggesting that it might be involved in l ocal movements. These studies provide the first structural insights in to a protein possessing GEF activity.