Human Taspase1 is essential for development and cancer by processing critical regulators, such as the mixed-lineage leukemia protein. Likewise, its ortholog, trithorax, is cleaved by Drosophila Taspase1 (dTaspase1), implementing a functional coevolution. To uncover novel mechanism regulating protease function, we performed a functional analysis of dTaspase1 and its comparisontothe human ortholog.dTaspase1contains an essential nucleophile threonine195, catalyzing cis cleavage into its a- and b-subunits. A cell-based assay combined with alanine scanning mutagenesis demonstrated that the target cleavage motif for dTaspase1 (Q3[F/I/L/M]2D1 G1'X2X3'v) differs significantly from the human ortholog (Q3[F,I,L,V]2D1G1'X2'X3'vD4'), predicting an enlarged degradome containing 70 substrates for Drosophila. In contrast to human Taspase1, dTaspase1 shows no discrete localization to the nucleus/nucleolus due to the lack of the importin-a/nucleophosmin1 interaction domain (NoLS) conserved in all vertebrates. Consequently, dTaspase1 interacts with neither the Drosophila nucleoplasmin-like protein nor human nucleophosmin1. The impact of localization on the protease's degradome was confirmed by demonstrating that dTaspase1 did not efficiently process nuclear substrates, such as upstream stimulatoryfactor2.However,geneticintroductionofthe NoLS into dTaspase1 restored its nucleolar localization, nucleophosmin1 interaction, and efficient cleavage of nuclearsubstrates.Wereportthatevolutionaryfunctional divergence separating vertebrates from invertebratescan be achieved for proteases by a transport/localizationregulated mechanism.
- Threonine aspartase