Glycosylation of nascent polypeptide chains in Aspergillus niger

Abstract

Polysomes were isolated from Aspergillus niger and were characterized on sucrose gradients in several ways. First, they were found to be susceptible to degradation by treatment with RNase or EDTA. Second, they were labeled after treating mycelia with short pulses of [ 3H]uridine or [ 3H]leucine prior to polysome isolation. Third, they were capable of stimulating incorporation of [ 3H]leucine into trichloroacetic acid-precipitable material in a chick reticulocyte cell-free protein-synthesizing system. When isolated [ 3H]leucine pulse-labeled polysomes were treated with either EDTA-RNase or puromycin, 80–90% of the radioactivity was released, indicating that only the nascent polypeptide chains were labeled. After exposing mycelia for 1 min to [ 14C]mannose, the polysomes were exclusively labeled, indicating that initial glycosylation takes place on nascent polypeptide chains. Preincubation of mycelia with 2-deoxyglucose followed by pulse-labeling with [ 3H]leucine and [ 14C]mannose showed that 2-deoxy- d-glucose inhibits both protein synthesis and glycosylation. However, similar preincubation with tunicamycin caused an 80% drop in [ 14C]mannose label in the polysomes, but only a 10–20% drop of [ 3H]leucine label, suggesting that glycosylation of nascent chains in A. niger involves an oligosaccharide-lipid intermediate, since it has been shown that tunicamycin inhibits the synthesis of such an intermediate. When isolated polysomes were placed into an in vitro glycosylating mixture containing Mn 2+, GDP-[ 14C]mannose, and smooth membranes from A. niger nascent chains were labeled. This reaction was shown to be dependent on addition of polysomes to the mixture and was not inhibited by 2-deoxy- d-glucose or tunicamycin. Both in vivo and in vitro glycosylated nascent chains were found to have about the same size range, and so it is suggested that in vitro no new oligosaccharide chains were synthesized, but preexisting chains were extended.