Divergent N-terminal sequences target an inducible testis deubiquitinatingenzyme to distinct subcellular structures

Ubiquitin-specific processing proteases (UBPs) presently form the largest e nzyme family in the ubiquitin system, characterized by a core region contai ning conserved motifs surrounded by divergent sequences, most commonly at t he N-terminal end. The functions of these divergent sequences remain unclea r. We identified two isoforms of a novel testis-specific UBP, UBP-t1 and UB P-t2, which contain identical core regions but distinct N termini, thereby permitting dissection of the functions of these two regions. Both isoforms were germ cell specific and developmentally regulated. Immunocytochemistry revealed that UBP-t1 was induced in step 16 to 19 spermatids while UBP-t2 w as expressed in step 18 to 19 spermatids, Immunoelectron microscopy showed that UBP-t1 was found in the nucleus while UBP-t2 was extranuclear and was found in residual bodies, For the first time, we show that the differential subcellular localization was due to the distinct N-terminal sequences. Whe n transfected into COS-7 cells, the core region was expressed throughout th e cell but the UBP-t1 and UBP-t2 isoforms were concentrated in the nucleus and the perinuclear region, respectively. Fusions of each N-terminal end wi th green fluorescent protein yielded the same subcellular localization as t he native proteins, indicating that the N-terminal ends were sufficient for determining differential localization. Interestingly, UBP-t2 colocalized w ith anti-gamma-tubulin immunoreactivity, indicating that like several other components of the ubiquitin system, a deubiquitinating enzyme is associate d with the centrosome. Regulated expression and alternative N termini can c onfer specificity of UBP function by restricting its temporal and spatial l oci of action.