HUMAN UBIQUITIN-ACTIVATING ENZYME, E1 - INDICATION OF POTENTIAL NUCLEAR AND CYTOPLASMIC SUBPOPULATIONS USING EPITOPE-TAGGED CDNA CONSTRUCTS

The ubiquitin-activating enzyme E1 catalyzes the first step in the ubi quitin conjugation pathway, Previously, we have cloned and sequenced t he cDNA for human E1. Expression of the E1 cDNA in the ts20 cell line, which harbors a thermolabile E1, abrogated the phenotypic defects ass ociated with this line. However, little is known of the cell biology o f the E1 protein or the nature of the E1 doublet. Thus, we constructed epitope-tagged E1 cDNAs in which the HA monoclonal antibody epitope t ag sequence (from influenza hemagglutinin and recognized by the 12CA5 monoclonal antibody) was fused to the amino terminus of E1. Because th e amino-terminal amino acid sequence of E1 is unknown, three construct s were made in which the HA tag was placed at each of the first three ATGs in the open reading frame (HA-1E1, HA-2E1, and HA-3E1). Western a nalysis of HeLa cells transfected with the constructs revealed that HA -1E1 closely comigrated with the upper band of the E1 doublet, and HA- 2E1 comigrated with the lower band of the E1 doublet; HA-3E1 appeared smaller than either of the E1 bands, Metabolic labeling with P-32 and immunoprecipitation with anti-HA antibody revealed that only the HA-1E 1 protein product is phosphorylated; polyclonal anti-E1 antibody showe d that only the upper band of the endogenous E1 doublet is phosphoryla ted, Each of the constructs was able to rescue the mutant phenotype of the ts20 cell line. Immunofluorescence studies showed that HA-2E1 and HA-3E1 were distributed in the cytoplasm with both negative and posit ive nuclei. This pattern of distribution has also been observed when i mmunostaining with a monoclonal antibody to E1 (1C5). However, the sta ining pattern associated with a polyclonal anti-E1 antibody (JJJ) is c haracterized by positive staining cytoplasm and nuclei in all cells. T he HA-1E1 construct exhibited apparently exclusive nuclear distributio n in HeLa cells. The difference between the staining patterns of the p olyclonal and monoclonal anti-E1 antibodies can be explained by the ex istence of two subpopulations of E1: one cytoplasmic and partially nuc lear, and one that is nuclear. Deletion of a small region at the amino terminus of the HA-1E1, including the basic sequence KKRR, transforme d its immunostaining pattern to that observed with HA-2E1.