Protoplasts of Saccharomyces strain 1016 took up [3H]glucosamine in the presence of an energy source; mannose was chosen to minimize randomization. It accumulated in the soluble intracellular pool primarily as UDP-N-acetyl[3H]glucosamine along with a small amount of [3H]glucosamine 6-phosphate. The antibiotic tunicamycin (TM) at 10 μg/ml did not affect the levels of these metabolites or inhibit the formation of the Nacetylglucosamine polymer, chitin, but did prevent the incorporation of [3H]glucosamine into mannan peptides and the synthesis of invertase. In vitro incorporation of [14C]mannose from GDP-[14C]mannose into mannan in a membrane preparation was not sensitive to 100 μg of TM/ml. TM appears to inhibit an N-acetylglucosaminyl transferase essential for glycoprotein biosynthesis. Binding of [3H]TM reflects its association with the plasma membrane fraction. This material could be recovered in an unaltered form by extraction with chloroform/methanol. If 0.2% phosphatidyl choline or phosphatidyl serine was added simultaneously with the [3H]TM, the binding of [3H]TM was greatly reduced, and the inhibitory effects of TM on protoplasts were prevented; however, addition of phospholipid 20 min later did not eliminate the inhibition, although about 80% of the bound [3H]TM was removed. TM interacts with lipophilic membrane components as well as inhibiting glycoprotein synthesis. © 1976.
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