EVIDENCE FOR SECRETION OF VACUOLAR ALPHA-MANNOSIDASE, CLASS-I CHITINASE, AND CLASS-I BETA-1,3-GLUCANASE IN SUSPENSION-CULTURES OF TOBACCO CELLS

We have investigated the possibility that vacuolar proteins can be sec reted into the medium of cultured cells of Nicotiana tabacum L. Time-c ourse and balance-sheet experiments showed that a large fraction, up t o ca. 19%, of vacuolar alpha-mannosidase (EC 3.2.1.24) and vacuolar cl ass I chitinase (EC 3.2.1.14) in suspension cultures accumulated in th e medium within one week after subculturing. This effect was most pron ounced in media containing 2,4-dichlorophenoxyacetic acid (2,4-D), Und er comparable conditions only a small fraction, 1.8-5.1% of the total protein and ca. 1% of malate dehydrogenase (EC 1.1.1.37), which is loc alized primarily in the mitochondria and cytoplasm, accumulated in the medium. Pulse-chase experiments showed that newly synthesized vacuola r class I isoforms of chitinase and beta-1,3-glucanase (EC 3.2.1.39) w ere released into the medium. Post-translational processing, but not t he release of these proteins, was delayed by the secretion inhibitor b refeldin A. Only forms of the proteins present in the vacuole, i.e. ma ture chitinase and pro-beta-1,3-glucanase and mature beta-1,3-glucanas e, were chased into the medium of tobacco cell-suspension cultures. Ou r results provide strong evidence that vacuolar alpha-mannosidase, chi tinase and beta-1.3-glucanase can be secreted into the medium. They al so suggest that secretion of chitinase and beta-1,3-glucanase might be via a novel pathway in which the proteins pass through the vacuolar c ompartment.