Immunolocalization of ubiquitin conjugates at Z-bands and intercalated discs of rat cardiomyocytes in vitro and in vivo.
Ubiquitin, a highly conserved 76-residue protein found in all eukaryotic cells, can be covalently bound to a wide variety of proteins in the nucleus, cytosol, cytoskeleton, and plasmalemma. This diversity of target proteins reflects a diversity of functions for ubiquitin conjugation. Previous studies have showed enhanced localization of ubiquitin conjugates to Z-bands of normal skeletal muscle and increased ubiquitination in atrophic muscles. These results have implicated a ubiquitin-mediated pathway in protein turnover and degradation in striated muscle. To investigate whether such a pathway might also exist in cardiac striated muscle, we used an affinity-purified polyclonal antibody (conjugate specific) and indirect immunofluorescence to localize ubiquitin conjugates in neonatal and adult rat cardiac myocytes both in vitro and in vivo. In both cultured myocytes and heart tissue, fluorescent ubiquitin conjugates were found in the nucleus as aggregates, in the cytoplasm in a striated pattern indicative of Z-bands, and in intercellular junctions at the intercalated discs between myocytes. Although the acceptor proteins and the physiological significance of ubiquitination at these locations are unknown, the targeting of ubiquitin to specific sites within the nucleus, myofibrils, and sarcolemma could provide a means for selective processing of individual components within these larger macromolecular assemblies, thus implying a regulatory role for ubiquitin conjugation in turnover or stability of proteins in the heart.
Hilenski
L L
LL
Department of Pathology (LLH,TKB), University of South Carolina, Columbia.
Terracio
L
L
Haas
A L
AL
Borg
T K
TK
D000818:Animals; D000906:Antibodies; D000911:Antibodies, Monoclonal; D002478:Cells, Cultured; D005455:Fluorescent Antibody Technique; D007365:Intercellular Junctions / Q000737:chemistry*; D009206:Myocardium / Q000737:chemistry*; D051381:Rats; D011919:Rats, Inbred Strains; D012508:Sarcolemma / Q000737:chemistry; D014452:Ubiquitins / Q000032:analysis*
D000906:Antibodies; D000911:Antibodies, Monoclonal; D014452:Ubiquitins