FUNCTIONAL-CHARACTERIZATION OF THE UBIQUITIN VARIANT ENCODED BY THE BACULOVIRUS AUTOGRAPHA-CALIFORNICA

The marked evolutionary conservation of ubiquitin is assumed to arise from constraints imposed by folding, stability, and interaction of the polypeptide with various components of the ATP,ubiquitin-dependent de gradative pathway. The present studies characterize the most divergent (75% identity) of the species-specific ubiquitin isoforms encoded as a late gene product of the baculovirus Autographa californica [Guarino , L. A. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 409-413]. Viral ubiqu itin supports 40% of the rate of ATP-dependent degradation exhibited b y eukaryotic ubiquitin. Inhibition of proteolysis correlated with a lo wer steady-state concentration of ubiquitin-conjugated degradative int ermediates. Rate studies revealed that viral ubiquitin exerts its effe ct at the step of isopeptide ligase-catalyzed (E3) ubiquitin conjugati on since viral and eukaryotic polypeptides are identical in their abil ities to support ATP-coupled activation by E1 and transthiolation to E 2 carrier proteins. Other studies demonstrated viral ubiquitin severel y attenuated the rate of K48-linked multiubiquitin chain formation in E3-independent conjugation catalyzed by recombinant yeast CDC34 or rab bit reticulocyte E2(32K) but not chain elongation of alternate linkage s formed by yeast RAD6 or human E2(EPF). The latter observations sugge st nonconserved positions on viral ubiquitin constitute recognition si gnals for K48-linked chain formation. Sequence comparison of species-s pecific ubiquitin isoforms indicates that nonconserved positions local ized to a defined region on the polypeptide surface distinct from the basic face required for E1 binding. These results suggest this novel u biquitin isoform may function in baculoviral replication to block dest ruction of a short-lived protein(s) by the host degradative pathway, t argeted through either E2-catalyzed K48-linked multiubiquitin chain fo rmation or general E3-mediated conjugation.