Regulation of cell wall invertase by a proteinaceous inhibitor

Suspension-cultured tobacco cells express a cell wall invertase (CWI) and a proteinaceous invertase inhibitor. Both proteins have been purified and characterized. A CWI cDNA clone has been isolated. The N-terminal protein sequence of the inhibitor has been determined showing high similarity with the N-terminal sequence of a tomato invertase inhibitor. A polyclonal antiserum has been raised against the denatured inhibitor protein. The observed changes of CWI activity during cell culture are partially the result of changes in gene expression, however, evidence has accumulated for further regulation by the inhibitor protein. In vivo regulation of CWI by the inhibitor is likely for the following reasons: (1) CWI inhibition is dependent on pH within the range relevant for the apoplasmic space, (2) divalent cations in the millimolar range affect CWI inhibition, (3) substrate protects CWI (but not vacuolar invertase) against inhibition, (4) CWI and inhibitor are co-expressed and co-localized in the apoplasmic space, (5) addition of inhibitor to intact cells causes substrate-protectable inhibition of CWI, (6) an in situ formation of tight complexes between CWI and inhibitor upon starvation of cells could be demonstrated. The results indicate that suspension-cultured tobacco cells are a suitable system to study the effects of an invertase inhibitor on CWI.