Tissue distribution of the “N-end rule” ubiquitin-conjugating enzyme, HR6, in the rat

Abstract

The conjugation of multiple ubiquitin molecules is required for recognition and degradation of a protein by the proteasome. The ubiquitination pathway responsible for the bulk of constitutive protein degradation targets proteins carrying basic or large hydrophobic amino acids at the N-terminus. In mammalian cells, this "N-end rule" pathway requires the ubiquitin-conjugating enzyme HR6. Until now, it has not been known which mammalian tissues and cell types predominantly utilize this pathway for degradation. Therefore, the distribution and intracellular localization of HR6 was determined by indirect immunofluorescence techniques and protein blotting of adult rat tissues. Intense immunoreactivity against HR6 was detected in various epithelia, muscle, testis, peripheral neurons, chromaffin cells and macrophages, whereas lower HR6 protein levels were found in the gut or in the kidney. Autonomic and sensory neurons, glandular cells and spermatocytes revealed prominent nuclear HR6 immunoreactivity. Plasma membrane labeling was observed in peripheral neurons, spermatocytes and skeletal muscle cells. Smooth muscle cells, macrophages, endothelial and epithelial cells exhibited primarily cytoplasmic staining. The clear differences in the regional and intracellular distribution of HR6 are suggestive for the involvement of N-end rule protein degradation in various physiological processes dependent on cell type and subcellular structure.