Limited proteolysis of yeast elongation factor 3 - Sequence and location of the subdomains

Elongation factor 3 (EF-3) is an ATPase essential for polypeptide chain syn thesis in a variety of yeasts and fungi. We used limited proteolysis to stu dy the organization of the subdomains of EF-3. Trypsinolysis of EF-3 at 30 degrees C resulted in the formation of three fragments with estimated molec ular masses of 90, 70, and 50 kDa. Yeast ribosomes protected EF-3 and the l arge fragments from further degradation. ATP exposed a new tryptic cleavage site and stabilized the 70- and 50-kDa fragments. The conformation of EF-3 as measured by fluorescence spectroscopy did not change upon ATP binding. Poly(G) stimulated proteolysis and quenched the intrinsic fluorescence of E F-3, Using gel mobility shift, we demonstrated a direct interaction between EF-3 and tRNA. Neither tRNA nor rRNA altered the tryptic cleavage pattern. The proteolytic products were sequenced by mass spectrometric analysis, EF -3 is blocked NH2-terminally by an acetylated serine. The 90-, 70-, and 50- kDa fragments are also blocked NH2-terminally, confirming their origin. The 50-kDa fragment (Ser(2)-Lys(443)) is the most stable domain in EF-3 with n o known function. The 70-kDa fragment (Ser(2)-Lys(668)) containing the firs t nucleotide-binding sequence motif forms the core ATP binding subdomain wi thin the 90-kDa domain. The primary ribosome binding site is located near t he loosely structured carboxyl-terminal end.