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

Abstract

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.

Getting membrane proteins on and off the shuttle bus between the endoplasmic reticulum and the Golgi complex

“…Although proteins with bulky lumenal domains might be excluded from crowded transport vesicles, as reported for glycosylphosphatidylinositol-anchored proteins (GPI-APs; Bhagatji et al, 2009), this possibility is less (Borgese et al, 2007); (ii) cytochrome P450 2C1 is anchored by an N-terminal 21-residue hydrophobic anchor to the ER membrane and does not possess known Sec24-interacting motifs; lengthening the N-terminal anchor by six residues results in its export from the ER (Szczesna-Skorupa and Kemper, 2000); (iii) the E protein of dengue virus comprises an N-terminal lumenal domain that is anchored to the ER bilayer through two contiguous membrane-spanning segments at the C-terminus. The E protein is retained in the ER, where assembly of viral particles occurs.…”

Clathrin-Independent Pathways of Endocytosis

“…A lipid-based sorting mechanism has been proposed because alterations in cholesterol and sphingolipid levels as well as perturbation of the nanoclustering of GPI-AP affects endocytosis via this pathway (Sharma et al 2004;Chadda et al 2007). The size of the extracellular moiety attached to the lipid tail may act as an additional determinant for inclusion into GEECs (Bhagatji et al 2009). Additionally, several transmembrane proteins such as CD44 and dysferlin are also selectively endocytosed via this pathway (Howes et al 2010a).…”

GPI-anchored protein organization and dynamics at the cell surface

“…A clear example, which links GPIanchored protein clustering at the plasma membrane and endocytosis, is in the context of MA104 cells where clustering of the FR, which is a GPI-anchored protein, is found necessary for ligand uptake ( 114 ). Synthetic GPI analogs, which have minimal structural similarity to endogenous GPI anchor, have been used in studies to dissect out the contribution of the glycan core in protein traffi cking ( 22 ). Similar studies would prove to be useful for understanding the structural and functional relationship of GPI-anchored proteins.…”

“…For the apical sorting of GPI-anchored protein oligomerization, N-glycosylation and raft association have been shown to be important ( 13 ); although the underlying mechanism for basolateral sorting of GPI-anchor proteins is not fully understood ( 21 ). Synthetic chemistry approaches have led to the development of modifi ed GPI analogs and, hence, allowed studying the traffi cking of analogs with modifi ed glycan cores, which showed that despite exhibiting a minor difference in the effi ciency of membrane incorporation, the traffi cking and endocytic fates of these analogs with differing glycan core does not show an appreciable change ( 22 ). However, there is compelling evidence that these analogs differ hugely in their diffusion properties, possibly due to their differential engagement with membrane molecules ( 6 ).…”