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Steric and not structure-specific factors dictate the endocytic mechanism of glycosylphosphatidylinositol-anchored proteins

¹ Department of Biochemistry, McGill University, Montreal, Quebec H3G 1Y6, Canada
² Department of Chemistry, St. Francis Xavier University, Antigonish, Nova Scotia B2G 2W5, Canada

Correspondence to John R. Silvius: john.silvius{at}mcgill.ca

Diverse glycosylphosphatidylinositol (GPI)-anchored proteins enter mammalian cells via the clathrin- and dynamin-independent, Arf1-regulated GPI-enriched early endosomal compartment/clathrin-independent carrier endocytic pathway. To characterize the determinants of GPI protein targeting to this pathway, we have used fluorescence microscopic analyses to compare the internalization of artificial lipid-anchored proteins, endogenous membrane proteins, and membrane lipid markers in Chinese hamster ovary cells. Soluble proteins, anchored to cell-inserted saturated or unsaturated phosphatidylethanolamine (PE)-polyethyleneglycols (PEGs), closely resemble the GPI-anchored folate receptor but differ markedly from the transferrin receptor, membrane lipid markers, and even protein-free PE-PEGs, both in their distribution in peripheral endocytic vesicles and in the manner in which their endocytic uptake responds to manipulations of cellular Arf1 or dynamin activity. These findings suggest that the distinctive endocytic targeting of GPI proteins requires neither biospecific recognition of their GPI anchors nor affinity for ordered-lipid microdomains but is determined by a more fundamental property, the steric bulk of the lipid-anchored protein.

Abbreviations used in this paper: CLIC, clathrin-independent carrier; DCC, dicyclohexylcarbodiimide; DHFR, dihydrofolate reductase; DIEA, diisopropylethylamine; DMF, dimethylformamide; GEEC, GPI-enriched early endosomal compartment; GPI, glycosylphosphatidylinositol; NHS, N-hydroxysuccinimide; PE, phosphatidylethanolamine; PEG, polyethyleneglycol; shRNA, small hairpin RNA; TFA, trifluoroacetic acid; Trt, trityl.

© 2009 Bhagatji et al.
This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.jcb.org/misc/terms.shtml). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).

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This article has been cited by other articles:

• Nichols, B. (2009). Endocytosis of lipid-anchored proteins: excluding GEECs from the crowd. JCB 186: 457-459 [Abstract] [Full Text]  

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