Abstract
Attachment of ubiquitin (Ub) to cell surface proteins serves as a signal for internalization via clathrin-mediated endocytosis (CME). How ubiquitinated membrane proteins engage the internalization apparatus remains unclear. The internalization apparatus contains proteins such as Epsin and Eps15, which bind Ub, potentially acting as adaptors for Ub-based internalization signals. Here, we show that additional components of the endocytic machinery including CALM, HIP1R, and Sla2 bind Ub via their N-terminal ANTH domain, a domain belonging to the superfamily of ENTH and VHS domains. Structural studies revealed that Ub binds with µM affinity to a unique C-terminal region within the ANTH domain not found in ENTH domains. Functional studies showed that combined loss of Ub-binding by ANTH-domain proteins and other Ub-binding domains within the yeast internalization apparatus caused defects in the Ub-dependent internalization of the GPCR Ste2 that was engineered to rely exclusively on Ub as an internalization signal. In contrast, these mutations had no effect on the internalization of Ste2 engineered to use an alternate Ub-independent internalization signal. These studies define new components of the internalization machinery that work collectively with Epsin and Eps15 to specify recognition of Ub as an internalization signal.
Highlights
Ubiquitination of membrane proteins dictates a variety of trafficking fates, usually ending in their degradation
Post-ER membrane proteins undergo a variety of Ubdependent sorting steps including transport to endosomes from the Golgi by GGAs, and MVB sorting by ESCRTs to incorporate ubiquitinated proteins into endosomal intralumenal vesicles that are delivered to the lysosome (Piper et al, 2014; Weeratunga et al, 2020)
Ubiquitination of membrane proteins is a signal for internalization via clathrin-mediated endocytosis (CME), a process largely conserved across eukaryotes (Kaksonen and Roux, 2018; Traub and Lukacs, 2007)
Summary
Ubiquitination of membrane proteins dictates a variety of trafficking fates, usually ending in their degradation. Post-ER membrane proteins undergo a variety of Ubdependent sorting steps including transport to endosomes from the Golgi by GGAs, and MVB sorting by ESCRTs to incorporate ubiquitinated proteins into endosomal intralumenal vesicles that are delivered to the lysosome (Piper et al, 2014; Weeratunga et al, 2020) Each of these sorting steps is mediated by an array of machinery that harbors multiple Ub-binding domains that recognize ubiquitinated proteins and, in some cases, help organize the processes they execute. The later stages of internalization are marked by the recruitment and polymerization of actin, leading to invagination and scission of the nascent endosome from the plasma membrane Coordination between these two phases is mediated in part by proteins such as Ent1/2 and Sla (Epsin and HIP1R in humans) that bridge PtdIns(4,5)P2 with the actin machinery. Internalization of Ste that used the NPFxD internalization signal was unaffected by the loss of multiple Ub-binding sites, implying these Ub-binding sites work collectively to recognize Ub as an internalization signal of cargo
Sign-in/Register to view highlights & summary 
Published Version
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call