Arf6-independent GPI-anchored Protein-enriched Early Endosomal Compartments Fuse with Sorting Endosomes via a Rab5/Phosphatidylinositol-3′-Kinase–dependent Machinery

Abstract

In the process of internalization of molecules from the extracellular milieu, a cell uses multiple endocytic pathways, consequently generating different endocytic vesicles. These primary endocytic vesicles are targeted to specific destinations inside the cell. Here, we show that GPI-anchored proteins are internalized by an Arf6-independent mechanism into GPI-anchored protein-enriched early endosomal compartments (GEECs). Internalized GPI-anchored proteins and the fluid phase are first visualized in GEECs that are acidic, primary endocytic structures, negative for early endosomal markers, Rab4, Rab5, and early endosome antigen (EEA)1. They subsequently acquire Rab5 and EEA1 before homotypic fusion with other GEECs, and heterotypic fusion with endosomes containing cargo from the clathrin-dependent endocytic pathway. Although, the formation of GEECs is unaffected by inhibition of Rab5 GTPase and phosphatidylinositol-3 -kinase (PI3K) activity, their fusion with sorting endosomes is dependent on both activities. Overexpression of Rab5 reverts PI3K inhibition of fusion, providing evidence that Rab5 effectors play important roles in heterotypic fusion between the dynamin-independent GEECs and clathrin-and dynamin-dependent sorting endosomes. INTRODUCTIONA cell uses multiple endocytic pathways for the uptake of molecules from the extracellular milieu (Mukherjee et al., 1997;Conner and Schmid, 2003). The better characterized pathways are mediated by clathrin-and caveolae-coated endocytic invaginations and require dynamin activity to be severed from the plasma membrane (Damke et al., 1994;Hinshaw and Schmid, 1995;Henley et al., 1998;Oh et al., 1998). There also seem to be one or more clathrin-independent pathways where dynamin is required, for example, the pathway for internalization of the interleukin (IL)-2 receptor subunits (Lamaze et al., 2001). In addition, there are a growing number of dynamin-independent endocytic processes that serve as internalization routes for a number of cell surface proteins, including lipid-linked proteins, such as GPI-anchored proteins (Lamaze and Schmid, 1995;Conner and Schmid, 2003;Nichols, 2003;Mayor and Riezman, 2004;Naslavsky et al., 2004;.GPI-anchored proteins are internalized via a dynamin, caveolin, and clathrin-independent pathway that is also responsible for the entry of a sizable fraction of the fluid phase in a number of cell types (Fivaz et al., 2002;Sabharanjak et al., 2002;Guha et al., 2003;. GPI-anchored proteins and the fluid phase enter the cell via tubular invaginations at the cell surface, into compartments termed GPI-anchored protein-enriched early endosomal compartments (GEECs) . At early times, these structures are largely devoid of cargo from the clathrin-dependent pathway. Besides a requirement for the small GTPase cdc42, very little is known about the molecular players that govern the formation and trafficking of GEECs. Much less is known about the detailed endocytic itinerary of these pathways (Lamaze and Schmid, 1995;Mayor and Riezman, 2004).After internalization...

Rapid Degradation of the Complement Regulator, CD59, by a Novel Inhibitor

“…Cells were then fixed and co-stained with EEA1 to assess whether CD59 reached early endosomes. As we and others have previously shown (41,42) antibody-bound internalized CD59 was predominantly found in early endosomes, as judged by its significant co-localization with endogenous EEA1 (Fig. 10A, pink structures in inset, quantified in C).…”

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