8.A.56 The Wntless Protein (Wls) Family 

wls, (srt, evi) of Drosophila is a segment polarity gene required for wingless (wg)-dependent patterning processes, acting in both wg-sending cells and wg-target cells. In non-neuronal cells, Wls directs wg secretion. The Wls traffic loop encompasses the Golgi, the cell surface, an endocytic compartment and a retrograde route leading back to the Golgi, and involves clathrin-mediated endocytosis and the retromer complex (a conserved protein complex consisting of Vps35 and Vps26) (Bänziger et al. 2006). In neuronal cells (the larval motorneuron NMJ), the wg signal moves across the synapse via the release of Wls-containing exosome-like vesicles (Korkut et al. 2009). Postsynaptic Wls is required for the trafficking of fz2 through the fz2-interacting protein Grip.  Sprinter (Srt) is required for secretioin of wingless (Wg) (Goodman et al. 2006) as Wg is retained by evi mutant cells (Bartscherer et al. 2006). 

Wnt proteins comprise a large class of secreted signaling molecules with key roles during embryonic development and throughout adult life. Porcupine and Wntless/Evi/Sprinter are required in Wnt-producing cells for the processing and secretion of many Wnt proteins, and secretion occurs independently of lipid modification (Ching et al. 2008).  All Wnts, except WntD, require Wls for secretion. All Wnts, except WntD, also contain a conserved serine residue (in Wg S239), which is essential for their functional and physical interaction with Wls. Finally, all Wnts except WntD, require the acyltransferase Porcupine for activity and for functionally interacting with Wls. Thus, Por-mediated lipidation of the S239-equivalent residue is essential for the interaction with, and secretion by, Wls (Herr and Basler 2012; Tang et al. 2012).  Together with the cargo receptor Evi/WIs, Wnts are transported through endosomal compartments onto exosomes, a process that requires the R-SNARE Ykt6 (Gross et al. 2012).  the Bro1-domain-containing protein Myopic (Mop) is indispensable for endosomal trafficking of Wg and Wls. Reductions in Mop leads to trapping of Wg and Wls in the early endosomes (Pradhan-Sundd and Verheyen 2014).

Xenopus laevis Wntless (XWntless) regulates the secretion of a specific Wnt ligand, XWnt4, and this regulation is required for eye development. The Retromer complex is required for XWntless recycling to regulate the XWnt4-mediated eye development. Inhibition of Retromer function by Vps35 morpholino (MO) results in various Wnt deficiency phenotypes, affecting mesoderm induction, gastrulation cell movements, neural induction, neural tube closure, and eye development (Kim et al. 2009). Disrupting the secretion of human Wnt5a also induced ER stress in mammalian cells, and a C-terminal KKVY-motif of Wg is required for its retrograde Golgi-to-ER transport, thus inducing ER stress. However, ER stress resulting from Wnt secretion impairment could be readily explained by its inability to leave the ER, and not resulting from Golgi-to-ER retrograde transport (Tang 2016).

Evi/Wntless plays a role in exporting Wnt proteins (Wolf and Boutros 2023). Intercellular communication by Wnt proteins governs many essential processes during development, tissue homeostasis and disease in all metazoans. Many context-dependent effects are initiated in the Wnt-producing cells and depend on the export of lipidated Wnt proteins. After lipid modification by the acyl-transferase, Porcupine, Wnt proteins bind their dedicated cargo protein Evi/Wntless for transport and secretion. Evi/Wntless and Porcupine are conserved transmembrane proteins, and their 3D structures are known. Wolf and Boutros 2023 summarized studies and structural data highlighting how Wnts are transported from the ER to the plasma membrane, and the role of SNX3-retromer during the recycling of its cargo receptor Evi/Wntless. The regulation of Wnt export through a post-translational mechanism and the importance of Wnt secretion for organ development and cancer are discussed.



Bänziger, C., D. Soldini, C. Schütt, P. Zipperlen, G. Hausmann, and K. Basler. (2006). Wntless, a conserved membrane protein dedicated to the secretion of Wnt proteins from signaling cells. Cell 125: 509-522.

Bartscherer, K., N. Pelte, D. Ingelfinger, and M. Boutros. (2006). Secretion of Wnt ligands requires Evi, a conserved transmembrane protein. Cell 125: 523-533.

Carette, J.E., C.P. Guimaraes, M. Varadarajan, A.S. Park, I. Wuethrich, A. Godarova, M. Kotecki, B.H. Cochran, E. Spooner, H.L. Ploegh, and T.R. Brummelkamp. (2009). Haploid genetic screens in human cells identify host factors used by pathogens. Science 326: 1231-1235.

Ching, W., H.C. Hang, and R. Nusse. (2008). Lipid-independent secretion of a Drosophila Wnt protein. J. Biol. Chem. 283: 17092-17098.

Franch-Marro, X., F. Wendler, S. Guidato, J. Griffith, A. Baena-Lopez, N. Itasaki, M.M. Maurice, and J.P. Vincent. (2008). Wingless secretion requires endosome-to-Golgi retrieval of Wntless/Evi/Sprinter by the retromer complex. Nat. Cell Biol. 10: 170-177.

Galli, L.M., N. Zebarjadi, L. Li, V.R. Lingappa, and L.W. Burrus. (2016). Divergent effects of Porcupine and Wntless on WNT1 trafficking, secretion, and signaling. Exp Cell Res 347: 171-183.

Gaspar, C.J., T. Gomes, J.C. Martins, M.N. Melo, C. Adrain, T.N. Cordeiro, and P.M. Domingos. (2023). Xport-A functions as a chaperone by stabilizing the first five transmembrane domains of rhodopsin-1. iScience 26: 108309.

Goodman, R.M., S. Thombre, Z. Firtina, D. Gray, D. Betts, J. Roebuck, E.P. Spana, and E.M. Selva. (2006). Sprinter: a novel transmembrane protein required for Wg secretion and signaling. Development 133: 4901-4911.

Gross, J.C., V. Chaudhary, K. Bartscherer, and M. Boutros. (2012). Active Wnt proteins are secreted on exosomes. Nat. Cell Biol. 14: 1036-1045.

Hausmann, G., C. Bänziger, and K. Basler. (2007). Helping Wingless take flight: how WNT proteins are secreted. Nat Rev Mol. Cell Biol. 8: 331-336.

Herr, P. and K. Basler. (2012). Porcupine-mediated lipidation is required for Wnt recognition by Wls. Dev Biol 361: 392-402.

Jeong, S.U., J.M. Park, S.Y. Yoon, H.S. Hwang, H. Go, D.M. Shin, H. Ju, C.O. Sung, J.L. Lee, G. Jeong, and Y.M. Cho. (2024). IFITM3-mediated activation of TRAF6/MAPK/AP-1 pathways induces acquired TKI resistance in clear cell renal cell carcinoma. Investig Clin Urol 65: 84-93.

Kim, H., S.M. Cheong, J. Ryu, H.J. Jung, E.H. Jho, and J.K. Han. (2009). Xenopus Wntless and the retromer complex cooperate to regulate XWnt4 secretion. Mol. Cell Biol. 29: 2118-2128.

Korkut, C., B. Ataman, P. Ramachandran, J. Ashley, R. Barria, N. Gherbesi, and V. Budnik. (2009). Trans-synaptic transmission of vesicular Wnt signals through Evi/Wntless. Cell 139: 393-404.

Pradhan-Sundd, T. and E.M. Verheyen. (2014). The role of Bro1- domain-containing protein Myopic in endosomal trafficking of Wnt/Wingless. Dev Biol 392: 93-107.

Tang, B.L. (2016). Are Wnts Retrogradely Transported to the ER? J Cell Physiol 231: 2315-2316.

Tang, X., Y. Wu, T.Y. Belenkaya, Q. Huang, L. Ray, J. Qu, and X. Lin. (2012). Roles of N-glycosylation and lipidation in Wg secretion and signaling. Dev Biol 364: 32-41.

Wolf, L. and M. Boutros. (2023). The role of Evi/Wntless in exporting Wnt proteins. Development 150:.

Xiao, Q., W. Rongfei, Z. Lingqiang, and H. Fuchu. (2015). The roles of signaling pathways in regulating kidney development. Yi Chuan 37: 1-7.


TC#NameOrganismal TypeExample

Wntless, Evenness interrupted or Sprinter (Wls; Evi; Srt) protein of 594 aas and 9 TMSs (Bartscherer et al. 2006).  Wls directs wg and wnt secretion (Franch-Marro et al. 2008; Hausmann et al. 2007). Wls forms a complex with Procupine (PORCN), and the two proteins have dissimilar functions in the overall secretion process  with Wls being the only one of these proteins to function directly in sercetion (Galli et al. 2016).  


Wls of Drosophila melanogaster (Fruit fly)


Wntless (Wls) protein of 541 aas and 8 TMSs in a 1 + 7 TMS arrangement.  Regulates Wnt protein sorting and secretion in a feedback regulatory mechanism. This reciprocal interaction plays a key role in the regulation of expression, subcellular location, binding and organelle-specific association of Wnt proteins (Xiao et al. 2015).


Wls of Homo sapiens


Uncharacterized protein of 538 aas and 9 probable TMSs in a 1 + 8 arrangement.

UP of Aethina tumida (small hive beetle)


Uncharacterized putative Wnt-binding factor required for Wnt secretion; pfam06664, of 534 aas and up to 11 TMSs.

UP of Nilaparvata lugens


TMEM181 of 612 aas and 8 TMSs in a 1 + 7 TMS arrangement. It mediates the action of cytolethal distending toxins (CDT), which are secreted by many pathogenic bacteria. Expression levels of TMEM181 are rate-limiting for intoxication (Carette et al. 2009).

TMEM181 of Homo sapiens


Rhodopsin-1, isoform A, light sensor of 547 aas and 8 TMSs in a 1 + 7 TMS arrangement (Gaspar et al. 2023).

Rhodopsin-1 of Drosophila melanogaster


TC#NameOrganismal TypeExample

Uncharacterized protein of 181 aas and 4 TMSs in a 2 + 2 arrangement.

UP of Lactobacillus oris


Uncharacterized protein of 177 aas and 4 TMSs in a 2 + 2 arrangement.

UP of Enterococcus saccharolyticus


Uncharacterized DUF3278 domain-containing protein of 169 aas and 4 TMSs in a 2 + 2 arrangement.

UP of Vagococcus fluviali


TC#NameOrganismal TypeExample

Uncharacterized molecular chaperone-like protein, GroL, of 616 aas with 4 or 5 C-terminal TMSs. If 4, they may be present in a 2 + 2 TMS arrangement like members of TC subfamily 8.A.56.2.

GroL of Pseudomonas syringae


RDD family domain-containing protein of 396 aas and 4 possible TMSs in a 2 + 2 TMS arrangement.

RDD domain protein of Alcanivorax sp. MD8A


J domain-containing protein of 426 aas and 2 C-terminal TMSs.

J domain protein of Bacillus cereus