Purification and characterization of Saccharomyces cerevisiae mitochondrial elongation factor Tu.
Yeast mitochondrial elongation factor Tu (EF-Tu) was purified 200-fold from a mitochondrial extract of Saccharomyces cerevisiae to yield a single polypeptide of Mr = approximately 47,000. The factor was detected by complementation with Escherichia coli elongation factor G and ribosomes in an in vitro phenylalanine polymerization reaction. Mitochondrial EF-Tu, like E. coli EF-Tu, catalyzes the binding of aminoacyl-tRNA to ribosomes and possesses an intrinsic GTP hydrolyzing activity which can be activated either by kirromycin or by ribosomes. Kinetic and binding analyses of the interactions of mitochondrial EF-Tu with guanine nucleotides yielded affinity constants for GTP and GDP of approximately 5 and 25 microM, respectively. The corresponding affinity constants for the E. coli factor are approximately 0.3 and 0.003 microM, respectively. In keeping with these observations, we found that purified mitochondrial EF-Tu, unlike E. coli EF-Tu, does not contain endogenously bound nucleotide and is not stabilized by GDP. In addition, we have been unable to detect a functional counterpart to E. coli EF-Ts in extracts of yeast mitochondria and E. coli EF-Ts did not detectably stimulate amino acid polymerization with mitochondrial EF-Tu or enhance the binding of guanine nucleotides to the factor. We conclude that while yeast mitochondrial EF-Tu is functionally analogous to and interchangeable with E. coli EF-Tu, its affinity for guanine nucleotides and interaction with EF-Ts are quite different from those of E. coli EF-Tu.
Rosenthal
L P
LP
Bodley
J W
JW
D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000032:analysis; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D007700:Kinetics; D008928:Mitochondria / Q000032:analysis*; D008970:Molecular Weight; D020653:Peptide Elongation Factor G; D010444:Peptide Elongation Factor Tu / Q000302:isolation & purification* / Q000378:metabolism; D010445:Peptide Elongation Factors / Q000378:metabolism; D010649:Phenylalanine / Q000378:metabolism; D011108:Polymers; D012346:RNA, Transfer, Amino Acyl / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000032:analysis*
D020653:Peptide Elongation Factor G; D010445:Peptide Elongation Factors; D011108:Polymers; D012346:RNA, Transfer, Amino Acyl; D006153:Guanosine Diphosphate; D010649:Phenylalanine; D006160:Guanosine Triphosphate; D010444:Peptide Elongation Factor Tu