The N-end rule proteolytic system in autophagy

Abstract

The N-end rule pathway is a cellular proteolytic system that utilizes specific N-terminal residues as degradation determinants, called N-degrons. N-degrons are recognized and bound by specific recognition components (N-recognins) that mediate polyubiquitination of low-abundance regulators and selective proteolysis through the proteasome. Our earlier work identified UBR4/p600 as one of the N-recognins that promotes N-degron-dependent proteasomal degradation. In this study, we show that UBR4 is associated with cellular cargoes destined to autophagic vacuoles and is degraded by the lysosome. UBR4 loss causes multiple misregulations in autophagic pathways, including an increased formation of LC3 puncta. UBR4-deficient mice die during embryogenesis primarily due to defective vascular development in the yolk sac (YS), wherein UBR4 is associated with a bulk lysosomal degradation system that absorbs maternal proteins from the YS cavity and digests them into amino acids. Our results suggest that UBR4 plays a role not only in selective proteolysis of short-lived regulators through the proteasome, but also bulk degradation through the lysosome. Here, we discuss a possible mechanism of UBR4 as a regulatory component in the delivery of cargoes destined to interact with the autophagic core machinery.

Keywords: N-recognin; UBR box; UBR4; angiogenesis; ubiquitin ligase; yolk sac.

Figure 1. The N-end rule pathway. In the mammalian N-end rule pathway, the tertiary destabilizing N-terminal residues Asn and Gln are deamidated by two distinct enzymes, NTAN1 and WDYHV1/NTAQ1 into Asp and Glu, respectively. The secondary destabilizing N-terminal residues Asp and Glu are arginylated by ATE1 R-transferase isoforms produced through alternative splicing of ATE1. N-terminal Cys is a tertiary destabilizing residue in mammals but not in S. cerevisiae. The destabilizing activity of N-terminal Cys requires its oxidation prior to arginylation by ATE1. C* denotes the oxidized N-terminal Cys residue, either Cys-sulfinic acid [CysO₂(H)] or Cys-sulfonic acid [CysO₃(H)]. N-terminal Arg together with other type 1 and type 2 residues are directly bound by a set of N-recognins characterized by the UBR box, including UBR1, UBR2, UBR4 and UBR5. UBR box proteins can target substrates through the recognition of internal degrons as well as N-degrons.

Figure 2. The structure and function of the visceral YS of murine embryos. (A) A cross-section of a mouse embryo at E9.5 with the YS intact. (B) An enlarged view of a dotted rectangle in (A). The visceral YS of early mouse embryos at E9.5 is composed of two developmentally distinct layers: an outer layer derived from the endoderm and an underlying layer derived from the mesoderm. The YS at E9.5 contains a constitutive, bulk protein degradation system that absorbs maternal proteins from the YS cavity and digests these foreign materials (as opposed to its own cellular constituents) using lysosomal hydrolases. The model illustrates that endocytosed maternal proteins are delivered to and digested by the lysosome through early endosomes (ee) and late endosomes (le). Lysosome-derived amino acids in the YS endoderm are supplied to the embryo via the vitelline circulation or used to synthesize paracrines that control vascular development in the mesodermal layer. AP, autophagosome; AMPH, amphisome; En, endothelial cells; Ly, lysosome; Nu, nucleus; Me, mesenchymal cell; He, hematopoietic cell.