Divergent N-terminal sequences target an inducible testis deubiquitinating enzyme to distinct subcellular structures.
Ubiquitin-specific processing proteases (UBPs) presently form the largest enzyme family in the ubiquitin system, characterized by a core region containing conserved motifs surrounded by divergent sequences, most commonly at the N-terminal end. The functions of these divergent sequences remain unclear. We identified two isoforms of a novel testis-specific UBP, UBP-t1 ... and UBP-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 was 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. When transfected into COS-7 cells, the core region was expressed throughout the 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 with green fluorescent protein yielded the same subcellular localization as the native proteins, indicating that the N-terminal ends were sufficient for determining differential localization. Interestingly, UBP-t2 colocalized with anti-gamma-tubulin immunoreactivity, indicating that like several other components of the ubiquitin system, a deubiquitinating enzyme is associated with the centrosome. Regulated expression and alternative N termini can confer specificity of UBP function by restricting its temporal and spatial loci of action.
Mesh Terms:
Age Factors, Amino Acid Sequence, Animals, Blotting, Northern, COS Cells, Cell Nucleus, Centrosome, DNA, Complementary, Endopeptidases, Green Fluorescent Proteins, Immunohistochemistry, Isoenzymes, Luminescent Proteins, Male, Microscopy, Immunoelectron, Molecular Sequence Data, RNA, Messenger, Rats, Rats, Sprague-Dawley, Recombinant Fusion Proteins, Spermatids, Testis, Time Factors, Tissue Distribution
Age Factors, Amino Acid Sequence, Animals, Blotting, Northern, COS Cells, Cell Nucleus, Centrosome, DNA, Complementary, Endopeptidases, Green Fluorescent Proteins, Immunohistochemistry, Isoenzymes, Luminescent Proteins, Male, Microscopy, Immunoelectron, Molecular Sequence Data, RNA, Messenger, Rats, Rats, Sprague-Dawley, Recombinant Fusion Proteins, Spermatids, Testis, Time Factors, Tissue Distribution
Mol. Cell. Biol.
Date: Sep. 01, 2000
PubMed ID: 10938131
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