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.
Lin
H
H
Department of Medicine, McGill University, Montreal, Canada.
Keriel
A
A
Morales
C R
CR
Bedard
N
N
Zhao
Q
Q
Hingamp
P
P
Lefrançois
S
S
Combaret
L
L
Wing
S S
SS
D000367:Age Factors; D000595:Amino Acid Sequence; D000818:Animals; D015152:Blotting, Northern; D019556:COS Cells; D002467:Cell Nucleus / Q000378:metabolism; D018385:Centrosome / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D010450:Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D049452:Green Fluorescent Proteins; D007150:Immunohistochemistry; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008164:Luminescent Proteins / Q000378:metabolism; D008297:Male; D016253:Microscopy, Immunoelectron; D008969:Molecular Sequence Data; D009124:Muscle Proteins; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013087:Spermatids / Q000378:metabolism*; D013737:Testis / Q000378:metabolism*; D013997:Time Factors; D014018:Tissue Distribution; D043222:Ubiquitin Thiolesterase
D018076:DNA, Complementary; D007527:Isoenzymes; D008164:Luminescent Proteins; D009124:Muscle Proteins; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D010450:Endopeptidases; D043222:Ubiquitin Thiolesterase; C464656:Usp2 protein, rat