Immunofluorescent localization of the ubiquitin-activating enzyme, E1, to the nucleus and cytoskeleton.

Ubiquitin, a 76-amino acid protein, is covalently attached to abnormal and short-lived proteins, thus marking them for ATP-dependent proteolysis in eukaryotic cells. Ubiquitin is found within the cytoplasm, nucleus, microvilli, autophagic vacuoles, and lysosomes. The ubiquitin-activating enzyme, E1, catalyzes the first step in ubiquitin conjugation. To date, very little is known about the subcellular distribution of this enzyme. We have utilized immunofluorescence and immunoblotting to examine the cellular distribution of E1 in several eukaryotic cell lines, including HeLa, smooth muscle A7r5, choriocarcinoma BeWo, Pt K1, and Chinese hamster ovary (CHO) E36. E1 was identified in both cytoplasmic and nuclear compartments in all cell lines examined. However, the relative abundance within these compartments differed markedly between the cell lines. Even within a single cell line, nuclear distribution was not uniform, and certain cells demonstrated an absence of nuclear staining. E1 resides predominantly within the nucleus in BeWo. In contrast, its distribution in CHO and Pt K1 cells is mainly cytoplasmic. Within the cytoplasm, three pools of E1 were identified by double-label immunofluorescence. The first of these colocalized with phalloidin, indicating association of E1 with actin filaments. A second cytoplasmic pool colocalized with tubulin and was predominantly perinuclear in its distribution. The third pool associated with intermediate filaments. This suggests that E1 is associated with all three components of the cytoskeleton. The distribution of E1 was unaltered in a mutant line of CHO E36 designated ts20, in which the E1 can be thermally inactivated. The variable distribution of E1 among cell lines, including its apparent cytoskeletal association, suggests pleiotropic functions of this enzyme and the ubiquitin-conjugating system.

Outcome of urgent percutaneous transluminal coronary angioplasty in acute myocardial infarction: comparison of single-vessel versus multivessel coronary artery disease.

Despite recent clinical trials of percutaneous transluminal coronary angioplasty (PTCA) in acute myocardial infarction, specific groups of patients that may benefit from adjunctive or alternative therapy have yet to be adequately characterized. The in-hospital outcome of 151 consecutive patients treated for acute myocardial infarction with urgent PTCA of the infarct-related artery was studied to identify a subgroup of patients at high risk. Patients were divided into two groups based on the angiographic presence of either single-vessel (n = 86) or multivessel (n = 65) coronary artery disease. Despite PTCA of only the infarct-related artery and similar baseline clinical characteristics such as age, peak serum creatine kinase concentration, left ventricular ejection fraction, and time from the onset of chest pain to arrival at the hospital, the group with multivessel disease had a lower rate of successful angioplasty (75% vs 92%, p < 0.005), with higher incidences of persistent total occlusion of the infarct-related artery (14% vs 3%, p < 0.02) and procedural complications during PTCA (28% vs 13%, p < or = 0.02), and were more likely to have multiple complications (12% vs 1%, p < 0.004). In addition, the group with multivessel disease had a higher rate of urgent (< or = 24 hours) coronary artery bypass graft surgery (13% vs 2%, p < 0.05) and a trend toward a higher in-hospital mortality rate (6% vs 1%, p < or = 0.17). By stepwise logistic regression, only the presence of single-vessel versus multivessel disease was predictive of PTCA success (p < 0.005).(ABSTRACT TRUNCATED AT 250 WORDS)

Ubiquitin-activating enzyme, E1, is associated with maturation of autophagic vacuoles.

The ubiquitin-activating enzyme, E1, is required for initiating a multi-step pathway for the covalent linkage of ubiquitin to target proteins. A CHO cell line containing a mutant thermolabile E1, ts20, has been shown to be defective in stress-induced degradation of proteins at restrictive temperature (Gropper et al., 1991. J. Biol. Chem. 266:3602-3610). Parental E36 cells responded to restrictive temperature by stimulating lysosome-mediated protein degradation twofold. Such a response was not observed in ts20 cells. The absence of accelerated degradation in these cells at 39.5 degrees C was accompanied by an accumulation of autolysosomes. The fractional volume of these degradative autophagic vacuoles was at least sixfold greater than that observed for either E36 cells at 30.5 degrees or 39.5 degrees C, or ts20 cells at 30.5 degrees C. These vacuoles were acidic and contained both acid phosphatase and cathepsin L, but, unlike the autolysosomes observed in E36 cells, ubiquitin-conjugated proteins were conspicuously absent. Combined, our results suggest that in ts20 cells, which are unable to generate ubiquitin-protein conjugates due to heat inactivation of E1, the formation and maturation of autophagosomes into autolysosomes is normal, but the conversion of autolysosomes into residual bodies is disrupted.

Immunoelectron microscopic localization of the ubiquitin-activating enzyme E1 in HepG2 cells.

As the first enzyme in the ubiquitin system the ubiquitin-activating enzyme E1 plays a pivotal role in all pathways of protein ubiquitination. In an effort to learn more about the cell biology of this pathway, we have purified the 110-kDa enzyme to homogeneity and generated a panel of distinct monoclonal antibodies to it. Using quantitative electron microscopic immunolocalization with these anti-E1 monoclonal antibodies, we find that E1 is abundant both within the cytoplasm and nucleus. Within the cytoplasm, E1 was found throughout the cytoplasmic volume as well as enriched along the cytoplasmic face of the rough endoplasmic reticulum and associated with the dense material along the desmosomal junctions. E1 was also found associated with the cytoplasmic surface of endosomal/lysosomal vacuoles. Interestingly, E1 was also found within the mitochondria. The lumen of rough endoplasmic reticulum, Golgi complex, endosomes, and lysosomes were negative. The specific localization of E1 to distinct subcellular organelles suggests that E1 may play multiple physiological roles within the cell.