Abstract
In the N-end rule pathway of protein degradation, the destabilizing activity of N-terminal Asp, Glu or (oxidized) Cys residues requires their conjugation to Arg, which is recognized directly by pathway's ubiquitin ligases. N-terminal arginylation is mediated by the Ate1 arginyltransferase, whose physiological substrates include the Rgs4, Rgs5 and Rgs16 regulators of G proteins. Here, we employed the Cre-lox technique to uncover new physiological functions of N-terminal arginylation in adult mice. We show that postnatal deletion of mouse Ate1 (its unconditional deletion is embryonic lethal) causes a rapid decrease of body weight and results in early death of ∼15% of Ate1-deficient mice. Despite being hyperphagic, the surviving Ate1-deficient mice contain little visceral fat. They also exhibit an increased metabolic rate, ectopic induction of the Ucp1 uncoupling protein in white fat, and are resistant to diet-induced obesity. In addition, Ate1-deficient mice have enlarged brains, an enhanced startle response, are strikingly hyperkinetic, and are prone to seizures and kyphosis. Ate1-deficient males are also infertile, owing to defects in Ate1−/− spermatocytes. The remarkably broad range of specific biological processes that are shown here to be perturbed by the loss of N-terminal arginylation will make possible the dissection of regulatory circuits that involve Ate1 and either its known substrates, such as Rgs4, Rgs5 and Rgs16, or those currently unknown.
Highlights
N-terminal arginylation of intracellular proteins by Arg-tRNAprotein transferase (R-transferase) is a part of the N-end rule pathway of protein degradation (Fig. 1A)
Our previous work has shown that the Ate1 promoter (PAte1) is bidirectional, expressing both Ate1 and an oppositely oriented gene termed Dfa, which overlaps with exon 1A of Ate1 (Fig. 1B) ([14]; C.S.B. and A.V., unpublished data)
Heterozygous Ate1+/2 mice were phenotypically similar to their wild-type (Ate1+/+) counterparts, we found that Ate1+/2 mice grew slightly but consistently slower than Ate1+/+ mice, and reached a lower average weight (Fig. 4B)
Summary
N-terminal arginylation of intracellular proteins by Arg-tRNAprotein transferase (R-transferase) is a part of the N-end rule pathway of protein degradation (Fig. 1A). In eukaryotes, this pathway is a part of the ubiquitin (Ub)-proteasome system. The main determinant of an N-degron is a destabilizing N-terminal residue of a substrate protein (Fig. 1A). ). Some of N-end rule substrates are produced by proteases that include MetAPs, separases, caspases and calpains. These and other nonprocessive proteases, which function as upstream components of the N-end rule pathway, have in common their ability to convert, through a cleavage, a pro-Ndegron into an N-degron. The present study expanded the earlier understanding of the Ate R-transferase (Fig. 1A) by making possible a postnatal inactivation of mouse Ate1. (Unconditional deletion of Ate results in embryonic lethality [10].) Described and discussed in Results is a large set of defects, some of them quite striking, in juvenile and adult mice upon the postnatal inactivation of Ate and the resulting
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