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Single-cell imaging of Wnt palmitoylation by the acyltransferase porcupine

Nature Chemical Biology volume 10, pages 61–68 (2014)Cite this article

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Abstract

Wnts are secreted palmitoylated glycoproteins that are important in embryonic development and human cancers. Here we report a method for imaging the palmitoylated form of Wnt proteins with subcellular resolution using clickable bioorthogonal fatty acids and in situ proximity ligation. Palmitoylated Wnt3a is visualized throughout the secretory pathway and trafficks to multivesicular bodies that act as export sites in secretory cells. We establish that glycosylation is not required for Wnt3a palmitoylation, which is necessary but not sufficient for Wnt3a secretion. Wnt3a is palmitoylated by fatty acids 13–16 carbons in length at Ser209 but not at Cys77, consistent with a slow turnover rate. We find that porcupine (PORCN) itself is palmitoylated, demonstrating what is to our knowledge the first example of palmitoylation of an MBOAT protein, and this modification partially regulates Wnt palmitoylation and signaling. Our data reveal the role of O-palmitoylation in Wnt signaling and suggest another layer of cellular control over PORCN function and Wnt secretion.

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Figure 1: Strategy for fluorescence imaging of palmitoylated Wnt3a proteins.
Figure 2: Palmitoylated Wnt3a is targeted to MVBs in secretory cells.
Figure 3: Wnt3a palmitoylation does not require glycosylation or regulate protein turnover.
Figure 4: O-palmitate bound to Wnt3a has slow turnover rate and regulates Wnt3a secretion.
Figure 5: Porcupine catalyzes the transfer of fatty acids 13–16 carbons in length onto Ser209 of Wnt3a.
Figure 6: Human PORCN is fatty acylated, and this modification regulates Wnt signaling.
Figure 7: Proposed model for Wnt3a palmitoylation, maturation and secretion.

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Acknowledgements

We would like to thank S. Scales for valuable comments on the manuscript and former members of the Hannoush lab for helpful discussions. We acknowledge use of microscopes at the Center for Advanced Light Microscopy at Genentech.

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  1. Department of Early Discovery Biochemistry, Genentech, Inc., San Francisco, California, USA

    Xinxin Gao & Rami N Hannoush

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X.G. and R.N.H. designed research. X.G. performed experiments. X.G. and R.N.H. analyzed data and wrote the paper. R.N.H. conceived of and guided the study.

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Correspondence to Rami N Hannoush.

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Gao, X., Hannoush, R. Single-cell imaging of Wnt palmitoylation by the acyltransferase porcupine. Nat Chem Biol 10, 61–68 (2014). https://doi.org/10.1038/nchembio.1392

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