In this article we report the identification of the sites which are involved in the binding of the GDP-exchange factor EF-1P and aminoacyl tRNA to the a-subunit of the eukaryotic elongation factor 1 (EF-1) from Artemiu. For this purpose the polypeptide chain of EF-la, having 461 amino acid residues, was proteolytically cleaved into large fragments by distinct proteases. Under well defined conditions, a mixture of two large fragments, free from intact EF-la and with molecular masses of 37 kDa and 43 kDa, was obtained. The 37-kDa and 43-kDa fragments comprise the residues 129 -461 and 69 -461, respectively. However, in aqueous solution and under non-denaturing conditions, the mixture still contained a short amino-terminal peptide, encompassing the residues 1 -36, that remained tightly bound. The ability of the mixture of the 37 + 43-kDa fragments, including this amino-terminal peptide 1 -36, to bind GDP or to facilitate aminoacyl tRNA binding to saltwashed ribosomes was severely reduced, compared to intact EF-la. However, both of these complexes were able to bind to the GDP-exchange-stimulating subunit EF-1P . A 30-kDa fragment, comprising the residues 1 -287, was generated after treatment of the protein with endoproteinase Glu-C. This fragment contained the complete guanine nucleotide binding pocket. Although it was able to bind GDP and to transport aminoacyl tRNA to the ribosome, no affinity towards EF-1P was observed. We propose that the guanine-nucleotide-exchange stimulation by EF-1P is induced through binding of this factor to the carboxy-terminal part of EF-la. As a result, a decreased susceptibility towards trypsin of the guanine-nucleotide-binding pocket of EF-la, especially in the region of its presumed effector loop is induced.The most abundant eukaryotic translational factor is elongation factor 1 (EF-I), which catalyzes, together with the translocation factor EF-2, the elongation cycle in the eukaryotic protein synthesis [I, 21. EF-1 contains four subunits (a, /J, y and 6). The elongation starts with the formation of a ternary complex between EF-la, aminoacyl tRNA and GTP. Subsequently, aminoacyl tRNA is attached to the mRNAribosome complex. This latter step requires hydrolysis of GTP [2]. The elongation rate is increased by two other subunits, EF-1P and EF-16, which both accelerate the exchange of GDP, bound to EF-la, for GTP [2, 31. EF-1P and EF-16 have been isolated in complex with EF-1y as heterodimers or heterotrimers (EF-Ifiy or EF-lPyG, respectively). Together with EF-1 a, these EF-IPy(6) complexes form high-moleclarmass aggregates, which have been described in many eukaryotic species [4 -81. Furthermore, these aggregates have been found associated with valyl tRNA synthetase [9, 101.
