Structural Basis of WLS/Evi-Mediated Wnt Transport and Secretion

Nygaard, Rie; Yu, Jia; Kim, Jonathan; Ross, Daniel R.; Parisi, Giacomo; Clarke, Oliver; Virshup, David M.; Mancia, Filippo

doi:10.1016/j.cell.2020.11.038

Cited by 46 publications

(58 citation statements)

References 93 publications

(135 reference statements)

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“…The exact ubiquitylation sites of EVI/WLS remain elusive, and mutational studies to investigate which residue is ubiquitylated by which enzyme are hindered by functional redundancy between the involved E2 enzymes and a large number of potential ubiquitin acceptor sites. Nevertheless, two lysine residues within the third intracellular loop of EVI/WLS are good candidates for ubiquitylation, as they are exposed and potentially easy to reach by interacting proteins ( Nygaard et al, 2021 ). Indeed, EVI/WLS–V5 overexpression constructs in which these lysines K410 and K419 were mutated to arginine showed reduced ubiquitylation after pulldown with K63-specific TUBEs ( Fig.…”

Section: Resultsmentioning

confidence: 99%

EVI/WLS function is regulated by ubiquitylation and is linked to ER-associated degradation by ERLIN2

Wolf

Lambert

Haenlin

et al. 2021

Journal of Cell Science

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WNT signalling is important for development in all metazoan animals and is associated with various human diseases. The Ubiquitin-Proteasome System (UPS) and regulatory ER-associated degradation (ERAD) have been implicated in the production of WNT proteins. Here, we investigated how the WNT secretory factor EVI/WLS is ubiquitinated, recognised by ERAD components and subsequently removed from the secretory pathway. We performed a focused, immunoblot-based RNAi screen for factors that influence EVI/WLS protein stability. We identified the VCP-binding proteins FAF2 and UBXN4 as novel interaction partners of EVI/WLS and showed that ERLIN2 links EVI/WLS to the ubiquitination machinery. Interestingly, we found in addition that EVI/WLS is ubiquitinated and degraded in cells irrespective of their level of WNT production. This K11, K48, and K63-linked ubiquitination is mediated by the E2 ubiquitin conjugating enzymes UBE2J2, UBE2K, and UBE2N, but independent of the E3 ligases HRD1/SYVN or GP78/AMFR. Taken together, our study identified factors that link the UPS to the WNT secretory pathway and provides mechanistic details on the fate of an endogenous substrate of regulatory ERAD in mammalian cells.

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Section: Resultsmentioning

confidence: 99%

EVI/WLS function is regulated by ubiquitylation and is linked to ER-associated degradation by ERLIN2

Wolf

Lambert

Haenlin

et al. 2021

Journal of Cell Science

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“…Previous research suggested Wnt hairpin-2 at interface 2 is not essential for WLS mediated Wnt secretion 41,43,44 , and we found interface 3 mainly involves limited hydrophobic interactions. Interface 2 and 3 may cooperate with each other to stabilize the interactions at interface 1, and help in later Wnts release and transfer to their receptors.…”

Section: Discussionmentioning

confidence: 43%

“…Hairpin-1 of Wnt3a carries the important palmitoleoylation site of Ser209, however, we could only observe extra density possible for PAM that is discontinuous from the density of Ser209 in the cryo-EM map. The possible PAM density lies between TM4 and TM5 with a similar position compared to that in the cryo-EM map of the just released WLS-Wnt8a structure 41 (Supplementary Fig. 7a).…”

Section: Pam Is Not Essential For Wnt3a-wls Associationmentioning

confidence: 69%

Cryo-EM structure of human Wntless in complex with Wnt3a

Zhong

Xiao

et al. 2021

Preprint

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Wntless (WLS), an evolutionarily conserved multi-pass transmembrane protein, is essential for secretion of Wnt proteins. Wnt-triggered signaling pathways control many crucial life events, whereas aberrant Wnt signaling is tightly associated with many human diseases including cancers. Here, we report the cryo-EM structure of human WLS in complex with Wnt3a, the most widely studied Wnt, at 3.8 Å resolution. The transmembrane domain of WLS bears a GPCR fold, with a conserved core cavity and lateral opening. A β-hairpin of Wnt3a containing a conserved palmitoleoylation site has extensive interactions with the WLS luminal domain, and the tip region is accommodated in the WLS cavity. The flexibility of Wnt3a loop/hairpin regions involved in the multiple binding sites indicates a possible sequential release mechanism when Wnts are transferred to their receptors. We found Wnt3a palmitoleoylation site mutant has similar binding capability with WLS compared with wild type Wnt3a, suggesting palmitoleoylation is not essential for Wnt-WLS association. Our findings provide important insights into the molecular mechanism of Wnt palmitoleoylation, secretion and signaling.

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“…Following the transport and modification of Wnt proteins in the ER, they are transferred to a cargo-receptor protein Wntless (also known as Sprinter/Evenness interrupted (Evi)/MIG-14/Gpr177, referred to as Evi hereafter), a multipass transmembrane protein which is an essential component of the Wnt secretory pathway (Bartscherer et al, 2006;Bänziger et al, 2006;Goodman et al, 2006). Recent structural analysis of the Wnt-Evi complex revealed that post palmitoylation, Wnt proteins are loaded on Evi through direct lateral transfer from Porcupine (Nygaard et al, 2021). Furthermore, the lipidation of Wnt protein is also essential for binding with Evi (Coombs et al, 2010;Herr and Basler, 2012).…”

Section: Synthesis and Intracellular Transport Of Wnt Proteinsmentioning

confidence: 99%

The Emerging Mechanisms of Wnt Secretion and Signaling in Development

Mehta

Hingole

Chaudhary

2021

Front. Cell Dev. Biol.

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Wnts are highly-conserved lipid-modified secreted proteins that activate multiple signaling pathways. These pathways regulate crucial processes during various stages of development and maintain tissue homeostasis in adults. One of the most fascinating aspects of Wnt protein is that despite being hydrophobic, they are known to travel several cell distances in the extracellular space. Research on Wnts in the past four decades has identified several factors and uncovered mechanisms regulating their expression, secretion, and mode of extracellular travel. More recently, analyses on the importance of Wnt protein gradients in the growth and patterning of developing tissues have recognized the complex interplay of signaling mechanisms that help in maintaining tissue homeostasis. This review aims to present an overview of the evidence for the various modes of Wnt protein secretion and signaling and discuss mechanisms providing precision and robustness to the developing tissues.

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Structural Basis of WLS/Evi-Mediated Wnt Transport and Secretion

Cited by 46 publications

References 93 publications

EVI/WLS function is regulated by ubiquitylation and is linked to ER-associated degradation by ERLIN2

EVI/WLS function is regulated by ubiquitylation and is linked to ER-associated degradation by ERLIN2

Cryo-EM structure of human Wntless in complex with Wnt3a

The Emerging Mechanisms of Wnt Secretion and Signaling in Development

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