Discriminatory interaction of purified eukaryotic initiation factors 4F plus 4A with the 5' ends of reovirus messenger RNAs.

The interaction of several reovirus mRNAs with cap-binding initiation factors has been investigated. Two quantitative experimental techniques have been applied to this question: (a) the rates of reaction of different mRNAs with tobacco acid pyrophosphatase and (b) the extent of cross-linking of different mRNAs to initiation factors in the presence and absence of ATP. The effects of ionic strength on these reactions have also been investigated. Our results demonstrate for the first time that the purified initiation factors interact differentially with purified reovirus mRNAs under competitive conditions and thus confirm earlier interpretations based on kinetic data. Comparison of the data from these studies with the translational behavior of the reovirus mRNAs, both in vitro and in vivo, has also led to specific predictions about features of these mRNAs that determine their competitive efficiencies. 1) Under ordinary ionic conditions, the steric accessibility of the m7G cap moiety of a reovirus mRNA appears to be a major determinant of its translation rate. 2) When the ionic strength is increased to supranormal levels, an additional feature, which may simply be the amount of secondary structure formed by sequences proximal to the cap, can become rate-limiting for several, but not all, of these mRNAs.

ATP-dependent unwinding of messenger RNA structure by eukaryotic initiation factors.

Interaction of protein synthesis initiation factors with mRNA has been studied in order to characterize early events in the eukaryotic translation pathway. Individual reovirus mRNAs labeled with 32P in the alpha position relative to the m7G cap and eukaryotic initiation factor (eIF)-4A, -4B, and -4F purified from rabbit reticulocytes were employed. It was found that eIF-4A causes a structural change in mRNA, as evidenced by a nuclease sensitivity test: addition of high concentrations of eIF-4A greatly increase the nuclease sensitivity of the mRNA, suggesting that this factor can melt or "unwind" mRNA structure. ATP is required for this reaction. At low concentrations of eIF-4A, addition of eIF-4B is required for maximal unwinding activity. Thus eIF-4B enhances eIF-4A activity. Addition of eIF-4F also makes the mRNA sensitive to nuclease indicating a similar unwinding role to that of eIF-4A. Stoichiometric comparisons indicate that eIF-4F is more than 20-fold more efficient than eIF-4A in catalyzing this reaction. The unwinding activity of eIF-4F is inhibited by m7GDP, while that of eIF-4A is not. This suggests that eIF-4A functions independent of the 5' cap structure. Our results also suggest that the unwinding activity of eIF-4F is located in the 46,000-dalton polypeptide of this complex, which has shown by others to be similar or identical to eIF-4A.

Maturation of alpha-mannosidase in Dictyostelium discoideum. Acquisition of endoglycosidase H resistance and sulfate.

alpha-Mannosidase from Dictyostelium discoideum is a heterogenous glycoprotein which is derived from a precursor as a result of proteolytic processing. Its oligosaccharides are phosphorylated and sulfated. We investigated the sulfation of the enzyme by means of pulse-chase labeling and specific immunoprecipitation followed by endoglycosidase H treatment and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The earliest detectable form of the precursor was shown to be glycosylated and sensitive to endoglycosidase H. With time some of its oligosaccharides were sulfated and became partially resistant to endoglycosidase H. In the same time period, the precursor was proteolytically cleaved, yielding four species with different molecular masses (46-58 X 10(3) daltons). When first generated each of these was sensitive to endoglycosidase H but with time the 54,000- and 58,000-dalton forms developed degrees of endoglycosidase H resistance. The fully mature cleaved forms all contained sulfate. Sulfate from pulse-labeled precursor could only be detected in two of the forms implying that sulfation of the others occurs either after precursor cleavage or before cleavage but subsequent to the pulse period. When secretion of precursor was triggered by starvation only the endoglycosidase H-resistant forms were secreted.

Interaction of Dictyostelium discoideum alpha-mannosidase with beef liver phosphomannosyl receptor. Effect of alkaline phosphatase treatment.

Both intracellular and secreted alpha-mannosidase from D. discoideum bind quantitatively and specifically to immobilized phosphomannosyl receptor from beef liver. Almost all the intracellular enzyme molecules retain tight binding capacity subsequent to exhaustive alkaline phosphatase treatment. This supports the idea that uncovered phosphates are not essential for optimal recognition by the phosphomannosyl receptor. However, approximately 50% of the extracellular enzyme was converted to a weak binding form by the same treatment.