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 synthesis in a variety of yeasts and fungi. We used limited proteolysis to study 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 molecular masses of 90, 70, and 50 kDa. Yeast ribosomes protected EF-3 and the large 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 EF-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 NH(2)-terminally by an acetylated serine. The 90-, 70-, and 50-kDa fragments are also blocked NH(2)-terminally, confirming their origin. The 50-kDa fragment (Ser(2)-Lys(443)) is the most stable domain in EF-3 with no known function. The 70-kDa fragment (Ser(2)-Lys(668)) containing the first nucleotide-binding sequence motif forms the core ATP binding subdomain within the 90-kDa domain. The primary ribosome binding site is located near the loosely structured carboxyl-terminal end.
Kambampati
R
R
Department of Pharmacy, University of Wisconsin, Madison, Wisconsin 53706, USA.
Pellegrino
C
C
Paiva
A
A
Huang
L
L
Mende-Mueller
L
L
Chakraburtty
K
K
D000595:Amino Acid Sequence; D005656:Fungal Proteins; D006868:Hydrolysis; D007700:Kinetics; D008969:Molecular Sequence Data; D010445:Peptide Elongation Factors / Q000737:chemistry / Q000378:metabolism*; D010449:Peptide Mapping; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D014357:Trypsin / Q000378:metabolism
D005656:Fungal Proteins; D010445:Peptide Elongation Factors; D029701:Saccharomyces cerevisiae Proteins; C111584:YEF3 protein, S cerevisiae; D014357:Trypsin