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Genome-wide analysis identifies a general requirement for polarity proteins in endocytic traffic

Nature Cell Biology volume 9, pages 1066–1073 (2007)Cite this article

Abstract

In a genome-wide RNA-mediated interference screen for genes required in membrane traffic — including endocytic uptake, recycling from endosomes to the plasma membrane, and secretion — we identified 168 candidate endocytosis regulators and 100 candidate secretion regulators. Many of these candidates are highly conserved among metazoans but have not been previously implicated in these processes. Among the positives from the screen, we identified PAR-3, PAR-6, PKC-3 and CDC-42, proteins that are well known for their importance in the generation of embryonic and epithelial-cell polarity. Further analysis showed that endocytic transport in Caenorhabditis elegans coelomocytes and human HeLa cells was also compromised after perturbation of CDC-42/Cdc42 or PAR-6/Par6 function, indicating a general requirement for these proteins in regulating endocytic traffic. Consistent with these results, we found that tagged CDC-42/Cdc42 is enriched on recycling endosomes in C. elegans and mammalian cells, suggesting a direct function in the regulation of transport.

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Figure 1: Distribution of positives within functional groups, and phenotypic profiling of yolk receptor (RME-2–GFP) localization.
Figure 2: Anterior PAR-complex components are required for efficient endocytosis.
Figure 3: Coelomocyte endocytosis and recycling endosome morphology are disrupted by loss of anterior PAR-complex components.
Figure 4: Altered trafficking of MHCI and transferrin in HeLa cells expressing dominant-negative Par6 or Cdc42, and association of HA–CDC-42 with recycling endosomes.

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Acknowledgements

We thank T. McGraw, J. Donaldson and I. Macara for important reagents; we also thank J. Donaldson and R. Weigert for their generous advice with the HeLa trafficking assays. We thank P. Schweinsberg, H. Dang and L. Shieh for technical assistance. This work was supported by NIH Grants GM67237 to B.D.G and GM65235 to H.F.

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Authors and Affiliations

  1. Department of Molecular Biology and Biochemistry, Rutgers University, Piscataway, 08854, NJ, USA

    Zita Balklava, Saumya Pant & Barth D. Grant

  2. Department of Molecular and Cellular Biology, University of Arizona, Tucson, 85721, Arizona, USA

    Hanna Fares

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  2. Saumya Pant
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Contributions

Z.B. participated in the experimental design, performed both screens, and performed and analysed endosome morphology and endocytic trafficking in worms and mammalian cells, and also wrote the paper. S.P. contributed to the secondary screen and quantification analysis. H.F. generated the transgenic C. elegans lines expressing coelomocyte endosome markers. B.D.G. designed the experiments and wrote the paper.

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Correspondence to Barth D. Grant.

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The authors declare no competing financial interests.

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Balklava, Z., Pant, S., Fares, H. et al. Genome-wide analysis identifies a general requirement for polarity proteins in endocytic traffic. Nat Cell Biol 9, 1066–1073 (2007). https://doi.org/10.1038/ncb1627

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