Construction and analysis of mouse strains lacking the ubiquitin ligase UBR1 (E3 alpha) of the N-end rule pathway

The N-end rule relates the in vivo half-life of a protein to the identity o f its N-terminal residue. In the yeast Saccharomyces cerevisiae, the UBR1-e ncoded ubiquitin ligase (E3) of the N-end rule pathway mediates the targeti ng of substrate proteins in part through binding to their destabilizing N-t erminal residues. The functions of the yeast N-end rule pathway include fid elity of chromosome segregation and the regulation of peptide import. Our p revious work described the cloning of cDNA and a gene encoding the 200-kDa mouse UBR1 (E3 alpha). Here we show that mouse UBR1, in the presence of a c ognate mouse ubiquitin-conjugating (E2) enzyme, can rescue the N-end rule p athway in ubr1 Delta S. cerevisiae. We also constructed UBR1(-/-) mouse str ains that lacked the UBR1 protein. UBR1(-/-) mice were viable and fertile b ut weighed significantly less than congenic +/+ mice. The decreased mass of UBR1(-/-) mice stemmed at least in part from smaller amounts of the skelet al muscle and adipose tissues. The skeletal muscle of UBR1(-/-) mice appare ntly lacked the N-end rule pathway and exhibited abnormal regulation of fat ty acid synthase upon starvation. By contrast, and despite the absence of t he UBR1 protein, UBR1(-/-) fibroblasts contained the N-end rule pathway. Th us, UBR1(-/-) mice are mosaics in regard to the activity of this pathway, o wing to differential expression of proteins that can substitute for the ubi quitin ligase UBR1 (E3 alpha). We consider these UBR1-like proteins and dis cuss the functions of the mammalian N-end rule pathway.