Abstract
The N-end rule pathway is a proteolytic system in which single N-terminal amino acids of proteins act as a class of degrons (N-degrons) that determine the half-lives of proteins. We have previously identified a family of mammals N-recognins (termed UBR1, UBR2, UBR4/p600, and UBR5/EDD) whose conserved UBR boxes bind N-degrons to facilitate substrate ubiquitination and proteasomal degradation via the ubiquitin-proteasome system (UPS). Amongst these N-recognins, UBR1 and UBR2 mediate ubiquitination and proteolysis of short-lived regulators and misfolded proteins. Here, we characterized the null phenotypes of UBR4-deficient mice in which the UBR box of UBR4 was deleted. We show that the mutant mice die around embryonic days 9.5–10.5 (E9.5–E10.5) associated with abnormalities in various developmental processes such as neurogenesis and cardiovascular development. These developmental defects are significantly attributed to the inability to maintain cell integrity and adhesion, which significantly correlates to the severity of null phenotypes. UBR4-loss induces the depletion of many, but not all, proteins from the plasma membrane, suggesting that UBR4 is involved in proteome-wide turnover of cell surface proteins. Indeed, UBR4 is associated with and required to generate the multivesicular body (MVB) which transiently store endocytosed cell surface proteins before their targeting to autophagosomes and subsequently lysosomes. Our results suggest that the N-recognin UBR4 plays a role in the homeostasis of cell surface proteins and, thus, cell adhesion and integrity.
Highlights
The N-end rule pathway is a proteolytic system in which single N-terminal amino acids act as degradation determinants, called N-degrons [1,2,3]
No live mutants were retrieved at and beyond E11.5. These results suggest that UBR4 is indispensable for embryonic development at midgestation
These results suggest that UBR4 is required for the formation of multivesicular body (MVB) and proteolysis therein, at least in part providing a mechanism underlying the depletion of cell surface proteins in the absence of UBR4
Summary
The N-end rule pathway is a proteolytic system in which single N-terminal amino acids act as degradation determinants, called N-degrons [1,2,3]. Known N-degrons include Arg, Lys, His (type-1, positively charged), Trp, Phe, Tyr, Leu, and Ile (type-2, bulky hydrophobic) exposed at the N-termini of proteins in humans [4, 5] These N-terminal residues are selectively recognized by recognition components, called N-recognins [6]. UBR4 is a 570-kDa protein that binds both type-1 and type-2 N-degrons [6, 17, 18] This poorly characterized N-recognin does not have a known ubiquitination domain but is required for optimal degradation of a model N-end rule substrate as well as ubiquitination of huntingtin (HTT) proteins such as 73 poly-glutamine repeat-bearing mutant HTT (73Q-HTT) and 175Q-HTT [19]. The N-end rule substrates of p62 include N-terminally arginylated proteins such as molecular chaperones that reside in the endoplasmic reticulum (ER) and a number of cytosolic proteins [8, 29]
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