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 ubiquitin conjugation pathway. Previously, we have cloned and sequenced the cDNA for human E1. Expression of the E1 cDNA in the ts20 cell line, which harbors a thermolabile E1, abrogated the phenotypic defects associated with this line. However, little is known of the cell biology of the E1 protein or the nature of the E1 doublet. Thus, we constructed epitope-tagged E1 cDNAs in which the HA monoclonal antibody epitope tag sequence (from influenza hemagglutinin and recognized by the 12CA5 monoclonal antibody) was fused to the amino terminus of E1. Because the amino-terminal amino acid sequence of E1 is unknown, three constructs 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 analysis 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 32P and immunoprecipitation with anti-HA antibody revealed that only the HA-1E1 protein product is phosphorylated; polyclonal anti-E1 antibody showed that only the upper band of the endogenous E1 doublet is phosphorylated. 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 positive nuclei. This pattern of distribution has also been observed when immunostaining with a monoclonal antibody to E1 (1C5). However, the staining pattern associated with a polyclonal anti-E1 antibody (JJJ) is characterized by positive staining cytoplasm and nuclei in all cells. The HA-1E1 construct exhibited apparently exclusive nuclear distribution in HeLa cells. The difference between the staining patterns of the polyclonal and monoclonal anti-E1 antibodies can be explained by the existence of two subpopulations of E1: one cytoplasmic and partially nuclear, and one that is nuclear. Deletion of a small region at the amino terminus of the HA-1E1, including the basic sequence KKRR, transformed its immunostaining pattern to that observed with HA-2E1.
Handley-Gearhart
P M
PM
Edward Mallincrodt Department of Pediatrics, Washington University School of Medicine, St. Louis, Missouri.
Stephen
A G
AG
Trausch-Azar
J S
JS
Ciechanover
A
A
Schwartz
A L
AL
D000595:Amino Acid Sequence; D002467:Cell Nucleus / Q000201:enzymology*; D003593:Cytoplasm / Q000201:enzymology*; D018076:DNA, Complementary; D000939:Epitopes / Q000737:chemistry; D005455:Fluorescent Antibody Technique; D006367:HeLa Cells; D006801:Humans; D008025:Ligases / Q000032:analysis* / Q000276:immunology; D008969:Molecular Sequence Data; D009154:Mutation; D010641:Phenotype; D014162:Transfection; D044764:Ubiquitin-Activating Enzymes; D044767:Ubiquitin-Protein Ligases
D018076:DNA, Complementary; D000939:Epitopes; D044767:Ubiquitin-Protein Ligases; D008025:Ligases; D044764:Ubiquitin-Activating Enzymes