The Eps15 homology (EH) domain-containing protein, EHD1, has recently been ascribed a role in the recycling of receptors internalized by clathrin-mediated endocytosis. A subset of plasma membrane proteins can undergo internalization by a clathrin-independent pathway regulated by the small GTP-binding protein ADP-ribosylation factor 6 (Arf6). Here, we report that endogenous EHD proteins, as well as transgenic tagged EHD1, are associated with long, membranebound tubules containing Arf6. EHD1 appears to induce tubule formation, which requires nucleotide cycling on Arf6 and intact microtubules. Mutations in the N-terminal P-loop domain or deletion of the C-terminal EH domain of EHD1 prevent association of EHD1 with tubules or induction of tubule formation. The EHD1 tubules contain internalized major histocompatibility complex class I (MHC-I) molecules that normally traf®c through the Arf6 pathway. Recycling assays show that overexpression of EHD1 enhances MHC-I recycling. These observations suggest an additional function of EHD1 as a tubuleinducing factor in the Arf6 pathway for recycling of plasma membrane proteins internalized by clathrinindependent endocytosis. Keywords: Arf6/clathrin-independent/EHD1/MHC class I/recycling IntroductionEndocytic receptors such as the epidermal growth factor (EGF) receptor and the transferrin receptor contain signals within their cytoplasmic domains that mediate their rapid internalization from the plasma membrane (for reviews see Trowbridge et al., 1993;Bonifacino and Dell'Angelica, 1999). Internalization of these receptors is effected by a complex molecular machinery comprising clathrin and various clathrin-associated proteins (for reviews see Kirchhausen, 2000;Brodsky et al., 2001). These proteins assemble on the cytoplasmic face of the membrane to form a supramolecular complex known as a clathrin coat, which recruits the plasma membrane receptors by virtue of interactions with their endocytic signals. Clathrin-coated domains of the plasma membrane undergo invagination and eventually pinch off as clathrincoated vesicles. These vesicles carry the internalized receptors to the early endosomal system, from where some receptors (e.g. the EGF receptor) are targeted to late endosomes and then lysosomes for degradation, while others (e.g. the transferrin receptor) are recycled back to the plasma membrane via a morphologically distinct organelle known as the endosomal recycling compartment (ERC) (for a review see Gruenberg and Max®eld, 1995).Many other plasma membrane proteins lack conventional endocytic signals, but can nonetheless undergo internalization via clathrin-independent pathways (for a review see ). The mechanisms involved in clathrin-independent endocytosis are not well understood. Among the mechanisms that have been invoked for this type of internalization are uptake through non-coated invaginations of the membrane known as caveolae (Kurzchalia and Parton, 1999), endocytosis via lipid rafts , micropinocytosis (Lamaze and Schmid, 1995) and macropinocytosis (Hewlett et al., 1...

The trafficking of two plasma membrane (PM) proteins that lack clathrin internalization sequences, major histocompatibility complex class I (MHCI), and interleukin 2 receptor alpha subunit (Tac) was compared with that of PM proteins internalized via clathrin. MHCI and Tac were internalized into endosomes that were distinct from those containing clathrin cargo. At later times, a fraction of these internalized membranes were observed in Arf6-associated, tubular recycling endosomes whereas another fraction acquired early endosomal autoantigen 1 (EEA1) before fusion with the "classical" early endosomes containing the clathrin-dependent cargo, LDL. After convergence, cargo molecules from both pathways eventually arrived, in a Rab7-dependent manner, at late endosomes and were degraded. Expression of a constitutively active mutant of Arf6, Q67L, caused MHCI and Tac to accumulate in enlarged PIP(2)-enriched vacuoles, devoid of EEA1 and inhibited their fusion with clathrin cargo-containing endosomes and hence blocked degradation. By contrast, trafficking and degradation of clathrin-cargo was not affected. A similar block in transport of MHCI and Tac was reversibly induced by a PI3-kinase inhibitor, implying that inactivation of Arf6 and acquisition of PI3P are required for convergence of endosomes arising from these two pathways.

Cells infected with prions contain both prion protein isoforms cellular prion protein (PrP C ) and scrapie prion protein (PrP Sc ). PrPSc is formed posttranslationally through the pathological refolding of PrP C . In scrapieinfected ScN2a cells, the metabolism of both PrP isoforms involves cholesterol-dependent pathways. We show here that both PrP C and PrP Sc are attached to Triton X-100-insoluble, low-density complexes or "rafts." These complexes are sensitive to saponin and thus probably contain cholesterol. This finding suggests that the transformation PrP C 3 PrP Sc occurs within rafts. It also reveals the existence of rafts in late compartments of the endocytic pathway, where most PrP Sc resides. When Triton X-100 lysates of cells were incubated at 37°C prior to density analysis, PrP C was still found in buoyant complexes, although it now failed to sediment at high speed. This property was shared by another glycophosphatidyl inositol protein, Thy-1, and also by the raft resident GM1. In one ScN2a clone and in the brain of a Syrian hamster with scrapie, Triton X-100 extraction at 37°C permitted resolution of PrP C and PrP Sc into two distinct peaks of different densities. This suggests that there are two populations of PrP-containing rafts and may permit isolation of PrP C -specific rafts from those containing PrP Sc . Our findings reinforce the contention that rafts are involved in various aspects of PrP metabolism and in the "life cycle" of prions.Prions are unique proteinaceous pathogens that cause a series of fatal encephalopathies such as Creutzfeldt-Jakob disease of humans, scrapie of sheep, and bovine spongiform encephalopathy (1). Prions seem to propagate in the host by posttranslationally (2, 3) refolding a normal host protein, the cellular prion protein (PrP C ), 1 to an aberrant conformation (4, 5). The only known component of prions is the misfolded isoform of PrP C , the scrapie prion protein (PrP Sc ) (6, 7). Current evidence argues that direct interaction of PrP Sc with PrP C is a prerequisite for the transformation PrP C ϩ PrP Sc 3 2PrP Sc (8,9). PrP C is a phosphoinositol glycolipid (GPI)-anchored glycoprotein present on the surface of neurons and other cells (10,11). The PrP isoforms appear to be chemically identical (12) but differ in their conformation (4); PrP C contains ϳ40% ␣-helix and is devoid of ␤-sheet, whereas PrP Sc has more than 40% ␤-sheet (4, 13-16). The two PrP isoforms differ considerably in their properties; PrP C is readily soluble in most detergents and is completely degraded by proteases, whereas PrP Sc is insoluble in detergents, possesses a protease-resistant core termed PrP27-30, and polymerizes into amyloidic structures called prion rods (17,18). Since no isoform-specific PrP antibody has yet been developed, the disparate properties of PrP C and PrP Sc serve as the sole ways to differentiate experimentally between these proteins. The subcellular sites where PrP Sc is formed, and the trafficking pathways leading to these sites, remain largely unknown. Scrapie-infecte...