J. Biochem, 1982, Vol. 91, No. 1 191-200
© 1982 Japanese Biochemical Society
research-article |
The Role of Core-Oligosaccharide in Formation of an Active Acid Phosphatase and Its Secretion by Yeast Protoplasts1
Department of Agricultural Chemistry, Faculty of Agriculture, The University of Tokyo Yayoi, Bunkyo-ku, Tokyo 113
Tunicamycin (TM), an antibiotic that blocks glycosylation of glycoproteins by inhibiting the formation of dolichyl N-acetylglucosaminyl pyrophosphate was used to study the expression of active repressible acid phosphatase (r-APase) [EC 3.1.3.2 [EC] ] by yeast protoplasts. Secretion of active r-APase was completely inhibited by TM, however, neither accumulation of r-APase activity inside protoplasts nor inhibition of protein synthesis were observed on TM-treatment. The results led us to postulate that an enzymatically inactive form of nonglycosylated r-APase is accumulated in the membrane fraction on TM-treatment. Protoplasts of various r-APase mutants were radiolabeled with [S35]methionine in the presence or absence of TM, then membrane fractions were analyzed by sodium dodecyl sulfate-polyacrylamide-gel electrophoresis and an autoradiogram was prepared. In r-APase-producing proto plasts of the wild type strain, the membrane (100,000 × g sedimentable) fraction of TM-treated protoplasts contained, in addition to usual membrane proteins, three proteins with molecular weights of 59 K, 57K, and 55 K daltons. The three proteins are also formed in TM-treated protoplasts of the pho80 mutant constitutively form ing r-APase in the presence of P1. However, TM did not induce formation of these three proteins in the protoplasts of r-APase negative mutants (pho2, pho4, and pho8l), or in the protoplasts of temperature sensitive mutants (pho2ts, and pho4ts) at a nonpermissive temperature (35°C), or in TM-treated protoplasts of the wild type strain repressed by P1 The three proteins were located in the membrane fraction and not in the cytosol or outside of the protoplasts. The purified r-APase preparation deglycosylated by hydrogen fluoride (HF) treatment at 0°C for I h or by endo-ß-N-acetylglucosaminidase H (Endo H) treatment, yielded three proteins with molecular weights of 62 K, 60 K, 58 K or 60 K, 58 K, 56 K daltons, respec tively. We considered the purified r-APase preparation to be composed of three different molecules, which yielded three deglycosylated proteins. The difference of about 2,000 daltons in apparent molecular weight between the deglycosylated pro teins produced by HF and Endo H treatments is probably due to N-acetylgluco samine residues attached to asparagine residues in the r-APase proteins.
Our results indicate that the 59 K, 57 K, and 55 K proteins produced by TM- treatment are nonglycosylated forms of r-APase and that addition of core-oligosac charide is required for formation of active r-APase and its secretion by yeast proto plasts.
1 This study was supported in part by a research grant from the Ministry of Education, Science and Culture of Japan.