Construction and Analysis of Mouse Strains Lacking the Ubiquitin Ligase UBR1 (E3α) of the N-End Rule Pathway

Abstract

The N-end rule relates the in vivo half-life of a protein to the identity of its N-terminal residue. In the yeast Saccharomyces cerevisiae, the UBR1-encoded ubiquitin ligase (E3) of the N-end rule pathway mediates the targeting of substrate proteins in part through binding to their destabilizing N-terminal residues. The functions of the yeast N-end rule pathway include fidelity of chromosome segregation and the regulation of peptide import. Our previous work described the cloning of cDNA and a gene encoding the 200-kDa mouse UBR1 (E3α). Here we show that mouse UBR1, in the presence of a cognate mouse ubiquitin-conjugating (E2) enzyme, can rescue the N-end rule pathway in ubr1Δ S. cerevisiae. We also constructedUBR1^−/− mouse strains that lacked the UBR1 protein. UBR1^−/− mice were viable and fertile but weighed significantly less than congenic +/+ mice. The decreased mass of UBR1^−/− mice stemmed at least in part from smaller amounts of the skeletal muscle and adipose tissues. The skeletal muscle of UBR1^−/−mice apparently lacked the N-end rule pathway and exhibited abnormal regulation of fatty acid synthase upon starvation. By contrast, and despite the absence of the UBR1 protein, UBR1^−/− fibroblasts contained the N-end rule pathway. Thus, UBR1^−/− mice are mosaics in regard to the activity of this pathway, owing to differential expression of proteins that can substitute for the ubiquitin ligase UBR1 (E3α). We consider these UBR1-like proteins and discuss the functions of the mammalian N-end rule pathway.

ACKNOWLEDGMENTS

We are grateful to members of the Caltech Transgenic and Knockout Core Facility, especially to S. Pease, B. Kennedy, and A. Granados for their care of mice and expert technical help. We thank B. Kennedy for his assistance with mouse weighing, W. Rivas for help with the cardiac puncture procedure, and Greg Cope for assistance with the Northern analysis. We are grateful to H. P. Roest (Erasmus University, Rotterdam, The Netherlands) for a gift of plasmid 44.83 and to members of the Varshavsky laboratory for helpful discussions and support. We also thank T. Tasaki and F. Du for their comments on the manuscript.

A.V. gratefully acknowledges support by the Fellows Program of the International Institute for Advanced Studies (Kyoto, Japan). This work was supported by grants GM31530 and DK39520 from the National Institutes of Health to A.V.