Date Published: August 29, 2018
Publisher: Public Library of Science
Author(s): Sung Tae Kim, Yoon Jee Lee, Takafumi Tasaki, Joonsung Hwang, Min Jueng Kang, Eugene C. Yi, Bo Yeon Kim, Yong Tae Kwon, Kwang-Hyun Baek.
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.
The N-end rule pathway is a proteolytic system in which single N-terminal amino acids act as degradation determinants, called N-degrons [1–3]. 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 . The protein substrates carrying N-degrons can be degraded by either the UPS or the autophagy-lysosome system (hereafter autophagy) [7–9]. In the UPS, N-recognins induce ubiquitination and proteasomal degradation of the substrates [10, 11]. The mammalian genome encodes a family of N-recognins (UBR1, UBR2, UBR4/p600, and UBR5) that recognize type-1 N-degrons through their conserved UBR boxes [12, 13]. Amongst these N-recognins, UBR1 and UBR2 with a size of 200 kDa are single polypeptide E3 ligases that bind all type-1 and type-2 N-degrons [4, 14]. These RING finger E3 ligases mediate ubiquitination of short-lived regulators in the cytosol and nucleus as well as misfolded proteins [7, 15, 16]. 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 . UBR4 and UBR5 are key regulators that synthesize K11/K48-branched heterotypic ubiquitin chains, which are induced and destined for proteasomal degradation during proteotoxic stress . UBR5 is a 300 kDa E3 ligase that preferentially binds type-1 N-degrons [6, 20, 21]. The HECT domain protein mediates ubiquitination of short-lived proteins such as ATMIN . Besides the known N-recognins, the mammalian genome encodes at least three more UBR box proteins, UBR3, UBR6, and UBR7 [6, 23], whose functions remain largely unknown [24–26]. In addition to the UPS, N-degrons can induce proteolysis via autophagy. In the autophagic N-end rule pathway, the autophagic adaptor p62/SQSTM1/Sequestosome-1 binds type-1 and type-2 residues and deliver the substrates to autophagosomes, leading to lysosomal proteolysis [27, 28]. 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].
We have previously identified UBR4 as an N-recognin of the N-end rule pathway which binds type-1 and type-2 N-degrons of short-lived proteins to facilitate their ubiquitination and proteasomal degradation . In this study, we characterized the null phenotypes of UBR4-deficient mice. Our results show that UBR4-deficient embryos die associated with developmental defects in various processes such as neurogenesis and cardiovascular development (Fig 1). The mutant cells in various tissues do not exhibit tissue-specific morphology as if they cannot receive properly external paracrine signals (Figs 2A and 4A). The mutant cells are more separated from each other, which is in part attributed to the inability to maintain cell surface proteins and, thus, cell adhesion and integrity (Figs 4 and 5). Although the mechanism underlying the role of UBR4 in the homeostasis of cell surface proteins should be further investigated, one such mechanism may be relevant to our observation that the MVBs are not properly generated in the absence of UBR4 (Fig 7).