We report here on the molecular nature of an EMS-induced mutant, mn1-89, a leaky semidominant allele of the Miniature1 (Mn1) seed locus that encodes a seed-specific cell wall invertase, INCW2. The mn1-89 locus specifies normal levels of the Incw2 transcript but extremely low levels (about 6% of normal) of the protein and enzyme activity are expressed. Sequence analysis of Incw2 clones derived from the parental Mn1 and the mutant genotypes shows a C to T transition in the mn1-89 allele, leading to a single amino acid alteration (proline to leucine) near the C-terminus of the mutant INCW2 protein. Although this change is not in the catalytic domain, putative N-glycosylation sites, or the beta-fructosidase motif, it does lie in a motif that is well conserved among all plant invertases and related fructosyltransferases. On the basis of these genetic in planta data, we believe we have identified a proline residue in a hitherto unknown GPFG motif as critical for the stability of such proteins. The single base change (C to T) also leads to the elimination of a BglI restriction site in the mutant allele. Indeed, BglI restriction digests of genomic DNAs from mn1-89 and Mn1 genotypes show one and two fragments, respectively. Sequence analysis of RT-PCR-derived endosperm Incw clones from mn1-1 (the reference allele) seeds predict five amino acid substitutions relative to Mn1. Whether or not these sequences are encoded by the mn1-1 locus or another non-allelic Incw gene in the maize genome remains to be elucidated.